Immunogenic composition and method of developing a vaccine based on portions of the HIV matrix protein

ABSTRACT

The present invention relates to an immunogenic composition. More particularly, the present invention is a composition directed to eliciting an immune response to at least one covalent binding site of myristate on the HIV matrix protein. The present invention contemplates three categories of embodiments: protein or protein fragments, messenger RNA, or DNA/RNA. DNA/RNA compositions may be either naked or recombinant. The present invention further contemplates use with a variety of immune stimulants.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication Ser. No. 60/513,827 filed Oct. 23, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of virology and immunology.Particularly, but not exclusively, it relates to a method of inducing animmune response, and a substance based on the amino terminal end of thematrix protein (p17MA) and covalent binding site for myristate of theHIV virus for achieving the same.

2. Description of the Related Art

Introduction

Human Immunodeficiency Virus (HIV) is a retrovirus within the slow orLentivirus group, and is the cause of Acquired Immunodeficiency Syndrome(AIDS). Like many enveloped viruses, HIV fuses the viral and cellularmembrane, leading to infection and viral replication. Once it has fusedto a host cell, HIV transfers its genome across both the viral andcellular membranes into the host cell.

HIV uses its RNA as a template for making complementary viral DNA intarget cells through reverse transcription. Viral DNA can then integrateinto the DNA of an infected host. HIV infects cells having surface CD4,such as lymphocytes and macrophages, and destroys CD4 positive helper Tlymphocytes. (CD4 represents a Cluster of Differentiation Antigen no. 4that is part of both Th1 and Th2 cells.) Cell membrane molecules areused to differentiate leukocytes into various effector subsets. Ingeneral, four types of cell membrane molecules also known as cluster ofdifferentiation (CD) have been delineated. Type I and 11 aretransmembrane proteins (TPs) with opposite polarity crossing the plasmamembrane. Type III TPs crosses the plasma membrane several times andtherefore may form pores or channels. Type IV TPs are linked toglycosylphosphatidylinositol (GPI). CD4 is a type I transmembraneprotein expressed on a variety of cells including helper/inducer Tcells, monocytes, macrophages and antigen presenting cells.

This process relies in part on fusion protein, which is a component ofthe gp41 glycoprotein. The F protein structure is protease resistant.(Weissenhorn, Nature Vol. 387, pp. 426-430 (1997)) Using X-raycrystallography the three dimensional features of the F protein havebeen delineated.

The outer membrane proteins, gp41 and gp120, of the HIV virus arenon-covalently bound to each other. On the surface of the HIV viriongp120 and gp41 are assembled into a trimeric unit. Three molecules ofgp120 are assimilated with three gp41 molecules.

The gp120 molecule binds to a CD4 receptor on the surface of helper Tcells as well as macrophages and monocytes. This binding ischaracterized by a high affinity between the two molecules. High sialicacid content on the surface of the virus reduces the threshold bindingenergy needed to overcome repulsive electrostatic forces. (Sun, 2002)Membrane fusion of an HIV particle to a target host cell may thus beconsidered to involve the following steps:

-   -   1. interaction of viral bound CypA with host/cellular heparin.    -   2. viral attachment to target cell via CypA/heparin interaction.    -   3. gp120 binding to the CD4 molecule of the target cell. This        process requires coreceptor proteins also known as chemokine        receptors (CCR5 for T cells and CXCR4 for macrophages). The        virus then begins to fuse with the cell, producing structural or        conformational changes and exposing other receptors;    -   4. conformational three dimensional and/or tertiary structure        changes of the gp120 molecule exposing the fusion domain or F        protein of gp41;    -   5. dissociation of the gp120 from the gp41 molecule as a result        of the conformational change and the shedding of gp120;    -   6. folding of gp41 upon itself before piercing the plasma        membrane of the target cell    -   7. unfolding of the F protein; and    -   8. fusion of the membranes of the viral particle and host cell.        The insertion of the fusion peptide disrupts the integrity of        the lipids within the targeted host cell membrane. F protein        links the viral and the cellular membranes, such that upon        unfolding of the fusion protein, the plasma membrane of the        target cell and the viral membrane are spliced together.

The viral membrane of HIV is formed from the plasma membrane of aninfected host cell when the virus buds through the cell's membrane.Thus, the envelope includes some of the lipid and protein constituentsof the host cell. (Stoiber, 1996) (Stoiber, 1997) Some enveloped virusesuse spike proteins, etc., to mimic the host molecules in order to bindto target cell receptors and to enter other target cells. However, thesespikes can also be antigenic surfaces for immune system recognition andviral destruction. HIV protects itself against immune challenge (humoraland CD8 mediated) by the host. In addition to the variability ofconformational changes, gp120 provides other surface features thatdisguise it from immune detection and attack, such as a coating ofglycoproteins, covalently bound sialic acid residues, or stericocclusion. (Haurum, 1993) In short, HIV activates a variety of immuneresponses to its own advantage.

The core of the HIV virion functions as a command center. Inside an HIVvirion is a capsid composed of the viral protein p24 (CA). The capsidalso holds two single strands of RNA, each strand of which provides acopy of HIV's nine genes, which encode 15 proteins. Of the nine genes,three (gag, pol and env) are considered essential. Six additional genesare also found within the 9-kilobase pair RNA genome (vif, vpu, vpr,tat, rev, and nef). More specifically, the env gene holds theinformation or code for creation of gp160, which breaks down into gp120and gp41. Likewise the gag gene encodes the matrix (p17 or MA), capsid(p24 or CA), nucleocapsid (p9,p6 or NC). The pol gene provides thegenetic information for the virus to produce the reverse transcriptaseenzyme as well as the integrase enzyme and RNAseH enzyme. The other sixgenes are regulatory, and control the mechanisms of infection andreplication (tat, rev, nef, vif, vpr and vpu). Among other things, thenef gene holds information for efficient replication, while vpu holdsinformation regulating the release of new viral particles from theinfected host cell. Ultimately, in order for HIV to infect a targetcell, it must inject the HIV genetic material into the target cellscytoplasm.

As noted above, the nef gene is believed to aid efficient replication ofHIV. The creation of a new virus particle occurs at the host cell'smembrane. Nef appears to affect an infected cell's environment in a waythat optimizes replication. Viral proteins collect near the host cell'smembrane, bud out within the membrane, and break away. These proteinsare the three structural proteins (gp160, gp120, gp41) plus two otherinternal precursor polyproteins (Gag and the Gag-Pol). The Gag-Polprotein brings two strands of the positive RNA into the bud, whileprotease cuts itself free. After the virus has budded, protease cutsitself free and cuts up the rest of the proteins in Gag or Gag-Pol,releasing the various structural proteins and reverse transcriptase. Theviral proteins are not functional until they are separated by theprotease. Thus, protease is responsible for cleavage of Gag-Pol and thesmaller Gag polyprotein into structural proteins. Released proteins p24,p7 and p6 form a new capsid, while at the base of the lipid membrane isp17. In this process, gp160 breaks down into gp120 and gp41 by a hostenzyme.

The gag gene gives rise to a 55-kilodalton (kD) Gag precursor protein,also called p55 (Pr55^(gag)), which is expressed from the unsplicedviral messenger RNA (mRNA). During translation, the N terminus of thep55 is myristylated, triggering its association with the cytoplasmicaspect of cell membranes. The membrane-associated Gag polyproteinrecruits two copies of the viral genomic RNA along with other viral andcellular proteins that trigger the budding of the viral particles fromthe surface of an infected cell. After budding, p55 is cleaved by thevirally encoded protease (a product of the Pol gene), during the processof viral maturation into four smaller proteins designated MA (matrix orp17), CA (capsid or p24) and NC (nucleocapsid or p9 and p6.) (Cohen, P.T., et al., The AIDS Knowledge Base, 149 (1999)) Thus, the HIV corecontains four proteins, including p17. In summation, the HIV virus isencoded by three large genes encoding structural and enzymatic peptides(gag, pol and env) and six smaller regulatory genes (vif, vpu, vpr, tat,rev and nef). (Sande, Merle A., et al., The Medical Management of AIDS,Ch. 2 (6th ed. 1999))

Pr55^(gag) (the polypeptide encoded by the gag gene) is cleaved by viralprotease to generate four large and two small peptides. From N to Cterminus the following proteins are proteolytically produced: matrix(p17MA), capsid (p24CA), nucleocapsid (p7NC), and p6. The two smallpeptides include p2 located between p24CA and P7NC and p1 locatedbetween p7NC and p6. The matrix protein assembles inside the viral lipidbilayer and stabilizes it. (Zhou, Wenjun, et al., J. of Virology, pp8540-8548 (December 1996))

Two critical steps in HIV viral replication are controlled by the matrixprotein and the larger polyprotein precursor Pr55^(gag). (1) The nucleartargeting signal of the matrix protein. (2) The strong localizing signalto the cell plasma membrane of Pr55^(gag). (Lee, Young-Min, et al. J. ofVirology, pp 9061-9068 (November 1998))

The Pr55^(gag) localizing signal can differentiate between the membranesencompassing cellular organelles and the plasma membrane. The key to thespecificity of plasma membrane binding is conferred by a combination ofa basic residue rich domain (amino acids 17-31) and the presence of anN-terminal myristoyl moiety. The 14 carbon fatty acid myristate iscotranslationally attached to the N-terminus of the HIV Pr55^(gag). Thisplasma membrane targeting of Pr55^(gag) is essential for viral assemblyand budding. (Zhou, 1996)

The active nuclear transport of the preintegration complex of HIVdisease is controlled by two viral proteins, p17MA and Vpr. After fusionof the viral membrane to the target cell membrane the matrix proteinbecomes detached from the inner aspect of the lipid bilayer and severalmatrix molecules with viral RNA cross the nuclear membrane and enter thenucleoplasm. Therefore HIV-1 can infect non-dividing cells and is notdependent on the disintegration of the nuclear envelope which occursduring mitosis. Most viruses are not capable of infecting non-dividingcells. (Zhou, 1996)

The differential membrane binding of Pr55^(gag) and MA is due to amyristoyl switch. Myristate is covalently bound to the N-terminalglycine amino acid of the MA protein. (Ono, Akira, et al., J. ofVirology,” pp 4136-4144 (May 1999)) Upon cleavage of the Pr55^(gag) byviral protease the myristate moiety inserts into a preexisting cavity ofthe MA molecule. This change in the three dimensional structure of theMA molecule occurs as a result of altered conformation in amino acids9-11 (serine, glycine, glycine) Therefore the MA molecule has lesselectrostatic force holding it to the lipid bilayer than its largerprecursor Pr55^(gag). Disruption of the viral plasma membrane uponfusion to a target cell destabilizes the matrix complex allowing fornuclear localization to occur. The flipping of the myristate moiety inand out of a protected cleft is known as the myristoyl switch and isfound in other proteins including ADP ribosylation factor (ARF),recoverin and c-Abl. Current estimates of myristoylated proteins in thehuman genome approximate 0.5%. (Resh, Marilyn D., “A myristoyl switchregulates membrane binding of HIV-1 Gag,” Proc. Natl. Acad. Sci., Vol101 (2) 417-418 (Jan. 13, 2004))

Myristylated proteins are covalently attached to myristic acid[tetradecanoic acid CH₃(CH₂)₁₂COOH]. Myristic acid is a carboxylic acid.Carboxylic acid molecules are polar and like alcohol molecules can formhydrogen bonds with each other and with other kinds of molecules.Myristic acid is virtually insoluble in water but is highly soluble inlipids explaining in part the plasma membrane localizing signal inherentin the MA molecule.

Fatty acid components of proteins can serve as regulated targetingdevices. (Tedeschi, Henry, Cell Physiology Molecular Dynamics, ch. 4(2003)) The carboxyl end of several myristylated proteins providehydrophobic anchors used in protein targeting and in signaltransduction. The fatty acid site may attach via hydrophobicinteractions to the phospholipid bilayer. Myristic acid is addedcotranslationally (while the protein is being synthesized) on terminalglycine amino acids.

The MA polypeptide (p17) is derived from the N-terminal, myristylatedend of p55. Most MA molecules remain attached to the inner surface ofthe virion lipid bilayer, stabilizing the particle. A subset of MA isrecruited inside the deeper layers of the virion where it becomes partof the complex which escorts the viral DNA to the nucleus after fusionof the viral and host membranes have occurred. These MA moleculesfacilitate nuclear transport of the viral genome because a karyophilicsignal on MA is recognized by the cellular nuclear import machinery.This is important because this allows HIV to infect non-dividing cells,such as macrophages, which is an unusual property for a retrovirus.(Cohen, P. T., et al., The AIDS Knowledge Base 149 (1999))

Most HIV vaccines, however, use portions of envelope glycoproteins(gp160, gp120, and gp41) in an attempt to induce production ofneutralizing antibodies against the envelope spikes of the virus.(Johnston, et al., 2001) Some have been successful in producing hightiters of neutralizing antibodies. The thought behind this approach isthat the antibodies that bind to these glycoproteins would neutralizethe virus and prevent infection. A functioning immune system could thenactivate the complement system, which would cascade to lysis and destroythe virus. The complement system is a series of circulating proteinsthat “complements” the role of antibodies. The components of thecomplement system are activated in sequence or turn, which is thecomplement cascade. The conclusion of complement is a protein complex,the Membrane Attack Complex (MAC) that seeks to attach to an invadingorganism's surface and to destroy it by puncturing its cell membrane.

Immune Response

Thus, a primary effect of HIV is to deplete the CD4 T+ cells, whichlowers overall immune activity. As described above, HIV infectioncenters on CD4 T+ cells, but it also infects B cells, blood platelets,endothelial cells, epithelial cells, macrophages, etc. As CD4 T+ cellsare depleted, the B cell response becomes deregulated.Hypergammaglobulinemia with ineffective antibodies characterizes HIVprogression. Further, cytotoxic CD8 T cells are rendered incompetent andare unable to recognize and attack viral infection. This is due in partto transfection of uninfected CD8 cells with the tat proteinmanufactured in infected CD4 cells.

The CD4 T+ helper (Th) cells produce cytokines and can be grouped intoeither Th1 cells or Th2 cells. The Th1 cells promote cell-mediatedimmunity while Th2 cells induce humoral immunity. The cytokines arechemical messengers or protein attractants that regulate immunologicresponses. The depletion of CD4+ helper cells in HIV disease results inreduced synthesis of certain cytokines and enhanced synthesis of others.Cytokine disregulation depresses the activity of the natural killercells and macrophages. Further, the loss of interleukin-2 slows theclonal expansion and activation of mature T cells.

Different viral traits augment or diminish cell mediated and humoralresponse. In some strains and phases of progression, HIV may becharacterized as a failure of Th1 response, accompanied by overactivebut ineffective Th2 response. The balance between Th1 and Th2 immuneresponse appears to depend in part on the HIV strain(s) and in part onthe genetic milieu of the infected animal. For example, long termnonprogressors mount an effective Th1 response to HIV disease.(Pantaleo, 1995)

An immunogenic compound directed to creating a balanced immune responseand strengthening or reinforcing the type of immune response suppressedby a particular virus would be of value. (Hogan, 2001)

Cellular Response

HIV appears to trigger an initially strong cellular immune response thatis not maintained over time and ultimately fails to control theinfection. (McMichael, 2001)

CD8 cytotoxic T-cells (Tc) recognize a cell presenting a foreign antigenby MHC (Major Histocompatibility Complex) class I molecules on thesurface, and attack it. CD4 helper cells (Th) stimulate macrophages thathave ingested a viral microbe to kill the microbe. The cytokines orinterleukins produced by the CD4 cells determine in part whether theimmunologic response to a pathogen is primarily TH1 or TH2 driven. Insome infections CD4 cells produce interleukin-4 and interleukin-5, whichselect for B-cells. B cells present antigen complexed with MHC class IImolecules. In other infections CD4 cells produce IL-2 which select forcytotoxic T cells. This division or restriction of functions inrecognizing antigens is sometimes referred to as MHC restriction. MHCclass I generally presents endogenously synthesized antigens, such asviral proteins, while MHC class II generally presents extracellularmicroorganisms or antigens such as bacterial or viral proteins whichhave been phagocytosed by antigen presenting cells. The antigenpresenting cells then bind the antigen with MHCII protein on itssurface. The CD4 cell interacts with this antigen through its T cellreceptor and becomes activated. This contributes to the ineffectivenessof inactivated vaccines to produce Tc cytotoxic response. (Levinson,2002)

As noted above, T cells mediate cellular response. The antigenpresenting cells, along with MHC molecules (or Human LeukocyteAntigen—HLA) present peptide portions of HIV antigens (or epitopes) totheir respective T cells, triggering T cell response. The type ofepitope presented to a T cell depends on the type of HLA molecule (e.g.,HLA A, B, C, DR, DQ, DP) and the amino acid in the peptides. Geneticlimitations in HLA molecules or mutant epitopes may lead to epitopeescape and HIV persistence. (McMichael, 2001) As noted above, Th cellsproduce cytokines for general (i.e., Th 1 and Th2) immune response, butin the case of HIV this is suppressed by infection of the Th cells. HIVspecific Th cells that respond to HIV antigens are eventually infectedand destroyed or suppressed. This leads to a secondary effect oncytotoxic T cells. Cytotoxic T cells demonstrate a variety of antiviralactivities (such as the production of performs, granzymes, FasL andcytokines), after recognizing and attacking foreign antigens on infectedcells that are bound by MHC class I molecules. HIV can reduce theexpression of HLA class I molecules in infected cells, reducing theability of cytotoxic T cells to recognize and attack the infected Thcells. Further, the infection and depletion of Th cells disrupt theability of cytotoxic T cells to mature and to address mutant virions.(McMichael, 2001) Typically, in a viral infection the cytotoxic T cellseliminate or suppress the virus. But HIV counters cellular immuneresponse by infecting immune cells and impairing the response of Thcells and cytotoxic T cells.

Thus, an immunogenic compound that stimulated Th 1 activity wouldpromote favorable immune response against HIV.

Humoral Response

The humoral arm of the immune system consists of B cells that, uponstimulation, differentiate into antibody producing plasma cells. Thefirst antibodies to appear are IgM, followed by IgG in blood, or IgA insecretory tissues. A major function of these antibodies is to protectagainst infectious disease and their toxins. Antibodies not onlyneutralize viruses and toxins, but also opsonize microorganisms.Opsonization is a process by which antibodies make viruses or bacteriamore easily ingested and destroyed by phagocytic cells. Phagocytic cellsinclude both polymorphonuclear neutrophils (PMNs) and tissuemacrophages. PMNs comprise about 60% of the leukocytes in the blood ofan uninfected patient. The number of PMNs and tissue macrophages mayincrease or decrease with certain infectious disorders. For example,typhoid fever is characterized by a decrease in the number of leukocytes(i.e., leukopenia). Both PMNs and macrophages phagocytose consumebacteria and viruses. PMNs do not present antigen to helper T cells,whereas macrophages and dendritic cells do.

Phagocytosis includes (1) migration, (2) ingestion, and (3) killing.Tissue cells in the infected area produce small polypeptides known aschemokines. The chemokines attract PMNs and macrophages to the site ofan infection. Then the bacteria are ingested by the invagination of thePMN cell membrane around the bacteria to form a vacuole or phagosome.This engulfment or opsonization is enhanced by the binding of IgGantibodies (opsonins) to the surface of the bacteria. The C3b componentof the complement system enhances opsonization. (Hoffman, R. HematologyBasic Principles and Practice Ch. 37 (3rd ed. 2000)) The cell membranesof PMNs and macrophage have receptors for C3b and the Fc portion of IgG.

With engulfment, a metabolic pathway known as the respiratory burst istriggered. As a result two microbicidal agents, the superoxide radicaland hydrogen peroxide are produced within the phagosomes. These highlyreactive compounds often called reactive oxygen intermediates aresynthesized by the following chemical reactions:O₂+e−−>O₂−2O₂−+2H+−>H₂O₂ (Hydrogen peroxide)+O₂

The first reaction reduces molecular oxygen to form the superoxideradical, which is a weak microbicide. The second reaction, which iscatalyzed by the enzyme superoxide dismutase within the phagosome,produces hydrogen peroxide. In general, hydrogen peroxide is a moreeffective microbicide than the superoxide radical. The respiratory burstalso produces nitrous oxide (NO), another microbicide. NO contains afree radical that participates in the oxidative killing of ingestedviruses and bacteria phagocytosed by neutrophils and macrophages. The NOsynthesis within the phagosome is catalyzed by the enzyme NO Synthase,which is induced by the process of phagocytosis.

The killing of the organism within the phagosome is a two step processthat consists of degranulation followed by the production ofhypochlorite ions, which is the most effective of the microbicidalagents. Two types of granules are found within the cytoplasm of theneutrophils or macrophages. These granules fuse with the phagosome toform a phagolysosome. The contents of the granules are then emptied.These granules are lysosomes that contain a variety of enzymes essentialto the killing and degradation. Two types of lysosomal granules, whichare differentiated by their size, have been identified. The largerlysosomal granule, which constitutes about 15% of the total, containsseveral enzymes including myeloperoxidase, lysozyme, and otherdegradative enzymes. The remaining 85% are smaller granules, whichcontain lactoferrin and other degradative enzymes, such as proteases,nucleases, and lipases. The actual killing or destruction ofmicroorganisms occurs by variety of mechanisms, some oxygen-dependentand some oxygen-independent. The most important oxygen-dependentmechanism is the production of the hypochlorite ion catalyzed bymyeloperoxidase:Cl⁻+H₂O₂−>ClO+H₂O

Antibodies are glycoproteins, composed of light (L) and heavy (H)polypeptide chains. The simplest antibody has a “Y” shape and consistsof four polypeptides: 2 H-chains and 2 L-chains. Disulfide bonds linkthe four chains. An individual antibody molecule will have identical H-and identical L-chains. L- and H-chains are subdivided into two regions:variable and constant. The regions have segments or domains, which arethree-dimensionally folded and repeating. An L-chain consists of onevariable (V1) and one constant (C1) domain. Most H chains consist of onevariable (VH) and three constant (CH) domains. The variable regions areresponsible for antigen (virus, bacteria, or toxin) binding. Theconstant regions encode several necessary biologic functions includingcomplement fixation and binding to cell surface receptors. Thecomplement binding site is located in the CH2 domain.

The variable regions of both L- and H-chains have three highly variable(or hypervariable) amino acids sequences at the amino-terminal portionthat makes up the antigen binding site. Only 5-10 amino acids in eachhypervariable region form this site. Antigen-antibody binding involveselectrostatic forces and van der Waals' forces. In addition, hydrogenand hydrophobic bonds are formed between the antigen and hyper-variableregions of the antibody. The specificity or “uniqueness” of eachantibody is in the hyper-variable region; the hyper-variable region isthe thumbprint of the antibody.

The amino-terminal portion of each L-chain participates in antigenbinding. The carboxy-terminal portion contributes to the Fc fragment.The Fc fragment (produced by proteolytic cleavage of the hinge region ofthe antibody molecule separating the antigen binding sites from the restof the molecule or the Fc fragment) expresses the biologic activities ofthe constant region, specifically complement fixation. The H-chains aredistinct for each of the five immunoglobulin classes. The heavy chainsof IgG, IgA, IgM, IgE and IgD are designated γ, α, μ, ε and δrespectively. The IgG immunoglobulin class opsonizes microorganisms;thus, this class of Ig (immunoglobulin) enhances phagocytosis. (Hoffman,Ronald, et al., Hematology Basic Principles & Practice, ch. 36 & 39 (3rded. 2000)) (Levinson, Warren, Medical Microbiology & Immunology, Ch. 59& 63 (7th ed. 2002)) Receptors for the γ H-chain of IgG are found on thesurface of PMNs and macrophages. IgM does not opsonize microorganismsdirectly because there are no receptors on the phagocyte surface for theμ H-chain. IgM does, however, activate complement, and the C3b proteincan opsonize because there are binding sites for C3b on the surface ofphagocytes. (Levinson, 2002) IgG and IgM, are able to initiatecomplement cascade. In fact, a single molecule of IgM can activatecomplement. Activation of complement by IgG requires two cross-linkedIgG molecules (IgG1, IgG2, or IgG3 subclasses, IgG4 has no complementactivity). A variety of non-immunologic molecules, such as bacterialendotoxin, can also activate the complement system directly.

The complement system consists of approximately twenty proteins that arenormally in serum. The term “complement” indicates how these proteinscomplement or augment other components in the immune system, such asantibodies and immunoglobulin. Complement cascade has three importantimmune effects: (1) lysis of microorganisms; (2) generation of mediatorsthat participate in inflammation and attract PMNs; and (3) opsonization.

Complement cascade occurs via one of three paths: (1) classic; (2)lectin; and (3) alternative. (Prodinger, Wm., et. al., FundamentalImmunology, Ch. 29 (1998)) These pathways are diagrammed in FIG. 1. Thedashed line shows that proteolytic cleavage of the molecule at the tipof the arrow has occurred. A line over a complex indicates that it isenzymatically active. Although the large fragment of C2 is sometimesinterchangeably labeled C2a or C2b, for convention, here small fragmentsare designated as “a,” and all large fragments as “b.” Hence, the C3convertase is C4b,2b. Note that proteases associated with themannose-binding lectin cleave C4 as well as C2. Each of these pathwaysleads to the creation of the Membrane Attack Complex (MAC).

With the antibody attached to a specific component of a virus orbacteria, the MAC is able to perforate the microorganism's protectivecover and allow blood plasma and electrolytes to enter themicroorganism, and at the same time provide a means for egress of themicroorganism's internal structural components.

In the classic pathway, antigen-antibody complexes activate C1 to form aprotease, which cleaves C2 and C4 to form a C4b,2b complex. C1 iscomposed of three proteins: C1q, C1r, and C1s. C1q is composed of 18polypeptides that bind to the Fc portion of IgG and IgM. Fc ismultivalent and can cross-link several immunoglobulin molecules. C1s isa proenzyme that is cleaved to form an active protease. Calcium isrequired as a cofactor in the activation of C1. Further, activation ofC1 requires multi-point attachment of at least two globular heads of C1qto the Fc domains of IgG and/or IgM. The changes induced in C1q onbinding multiple Fc immunoglobulins is transmitted to the C1rs subunits,resulting in proteolytic autoactivation of the C1r dimer, which thenproteolytically activates or cleaves C1s. As seen above, activated C1spossesses the catalytic site for proteolytic splicing of C4 and C2. Anenzyme complex, C4b,2b, is produced. This functions as a C3 convertase,which cleaves C3 molecules into two fragments, C3a and C3b. C3b forms acomplex with C4b and C2b, producing a new enzyme, (C4b,2b,3b) which is aC5 convertase.

In the lectin pathway, mannan-binding lectin (MBL, or mannose-bindingprotein) binds to the surface of microbes expressing mannan. MBP is aC-type lectin in plasma that has a structure similar to that of C1q, andbinds to C1q receptors enhancing phagocytosis. Mannose is an aldohexosefound on the surface of a variety of microorganisms. The first componentof the lectin pathway is designated mannose (or mannan) binding protein(MBP). A C-terminal carbohydrate recognition domain has affinity forN-acetylglucosamine and confers the capacity for MBP to directlyopsonize microorganisms expressing mannose-rich surface coats. In theblood, MBP circulates as a stable complex with a C1r-like proenzyme anda C1s-like proenzyme (designated MBP-associated serine protease, orMASP-1 and MASP-2 respectively). The MBP-MASP-1, MASP-2 complex binds tothe appropriate carbohydrate surface. This results in conformationalchange in the MBP protein which leads to auto-activation of MASP-1 byinternal peptide cleavage converting MASP-1 to an active serineprotease. Like C1r, active MASP-1 cleaves MASP-2 activating it. ActiveMASP-2 exhibits the capacity to proteolytically activate both C4 and C2to initiate assembly of the C4b,2b (C3 convertase) enzyme complex. Aswith the classic pathway, this leads to the production of C5 convertase.

In the alternative pathway many unrelated cell surface structures, suchas bacterial lipopolysaccharides (endotoxin), fungal cell walls, andviral envelopes, can initiate the process by binding to C3(H₂0) andfactor B. This complex is cleaved by a protease, factor D, to produceC3b,Bb, which acts as a C3 convertase to generate more C3b. In contrastto the sequential enzyme cascade of the classical pathway, thealternative pathway uses positive feedback; the principal activationproduct, C3b, acts as a cofactor for C3b,Bb, which is also responsiblefor its own production. Thus, the alternative pathway is continuouslyprimed for explosive C3 activation. The rate of C3 activation isgoverned by the stability of the C3b,Bb enzyme complex. Proteolysis offactor B by factor D produces a small fragment (Ba) and a large fragment(Bb). The larger Bb fragment combines with either C3(H₂0) or C3b.Through a catalytic site in Bb, the complex C3(H₂0),Bb canproteolytically convert C3 to C3a and C3b. Nascent C3b generated by thismechanism is capable of binding additional factor B. Therefore thealternative complement pathway has at least two positive feedback loopsenhancing the production of C3b. As shown in FIG. 1, this route alsoleads to the production of C5 convertase.

For each pathway the C5 convertase (C4b,2b,3b or C3b,Bb,C3b) cleaves C5into C5a and C5b. C5b binds to C6 and C7, to form a complex thatinteracts with C8 and C9, ultimately producing MAC (C5b,6,7,8,9).(Hoffman, 2000)

Regardless of which complement pathway is activated, the C3b complex isa central molecule for complement cascade. Immunologically C3b fulfillsthree roles:

-   -   1. sequential combination with other complement components to        generate C5 convertase, the enzyme that leads to production of        MAC (C5b,6, 7,8,9);    -   2. opsonization of microorganisms. Phagocytes have receptors for        C3b on their cell surface.    -   3. binding to its receptors on the surface of activated B cells,        which greatly enhances antibody production. (Parham, Peter, The        Immune System, ch. 7 (2nd ed. 2004))        The humoral response includes certain regulators of this system,        such as Complement Factor H, that are vulnerable to exploitation        by HIV. Any microorganism with the capacity to limit the        activity of complement cascade could theoretically protect        itself against the humoral arm of the immune system. (Stoiber,        Heribert, Role of Complement in the control of HIV dynamics and        pathogenis, Vaccine 21: S2/77-S2182 (2003)) Thus, the complement        cascade is an Achilles heel of the humoral arm.        HIV Interaction with Humoral Response

Retroviruses can activate the complement system in the absence ofantibodies. (Haurum, J., AIDS, Vol. 7(10), pp. 1307-13 (1993))Complement activation by HIV envelope glycoproteins has been found to bemediated by the binding of MBP to carbohydrates on natural envelopeprotein produced in virus-infected cells, as well as on glycosylatedrecombinant envelope proteins. (Haurum, John, AIDS, Vol. 7(10), pp.1307-13 (1993)) (Speth, C., Immunology Reviews, Vol. 157, pp. 49-67(1997)) Activation of the classical complement pathway and lectinpathway by retrovirus envelopes can be initiated by the binding of MBPto carbohydrate side chains of envelope glycoproteins. The transmembraneprotein of HIV-1, gp41, has been shown to be non-covalently associatedwith gp120. Complement component, C1q, also binds to gp41. In thecell-external part (ectodomain) of gp41, three sites (aa 526-538; aa601-613 and aa 625-655) bind both gp120 and C1q. Thus, C1q and gp120 areboth structurally and functionally homologous. The interaction betweengp41 and C1q is calcium dependent unlike the association of gp41 andgp120 which is calcium independent.

HIV triggers the classical and lectin pathway in an antibody-independentmanner which leads to the infection of complement receptor-positivecells by HIV. The binding of C1q to gp41 may facilitate infection indifferent ways. C1q binds directly to HIV-infected cells that are alsoinfected with HIV-1. C1q retains its ability to bind to the C1qreceptor, also known as the collectin receptor. Further, gp41 interactsdirectly with C1q anchored on the plasma membrane of macrophages. Inboth cases, HIV has the opportunity for C1q-mediated CD4 independentcontact with cells.

The homology of gp120 and C1q raises the possibility that gp120 mayinteract directly with the C1q receptor, and thereby facilitate theentry of HIV into macrophages in a CD4-independent manner. (Stoiber,Heribert, European Journal of Immunology, Vol 24, pp. 294-300 (1994))Antibodies to gp120 are able to cross react with C1q and may beresponsible, at least in part, for the significantly low C1qconcentration in HIV-1 patients. C1q is one of the factors responsiblefor the clearance of insoluble immune complexes, and its absence mightcontribute to the significantly high concentrations of insoluble immunecomplexes noted in individuals infected with HIV. (Procaccia, S., AIDSVol 5, p. 1441 (1991)) Hypocomplementemia which characterizes HIVdisease is correlated with HIV associated opportunistic infections andviral associated malignancies.

Regulators of complement activity can be found attached to plasmamembranes. Others circulate freely in human plasma and lymph. Manyregulators of complement activity (RCA) have been characterized andvirtually every step in all three pathways is subject to positive andnegative controls. Three enzymatic complexes (C3 convertases, C5convertases, MAC complex) are focal within the complement cascade andare subjected to multiple inhibitors or catalysts.

Several proteins that control the complement activation pathwayscirculate in plasma as freely soluble molecules, and can either controlC3 activation in the fluid phase or inhibit formation of MAC on cellsurfaces. Regulators of complement, such as Factor H andlow-molecular-weight Factor H-like proteins, have been shown to mediatethis function. Factor H interacts with gp120, enhancing syncytiumformation and soluble CD4 (sCD4) induced dissociation of the envelopeglycoprotein (env) complex. Factor H only binds activated gp120 after ithas engaged CD4, suggesting that the binding site is hidden within theenv complex, and becomes exposed only after interaction of gp120 withCD4 . (Pinter, C., AIDS Research in Human Retroviruses, Vol. 11, (1995))The gp120 molecule binds to the CD4 receptor on helper T cells. Thevirus then fuses with the T cell. The fusion domain is located on gp41.Upon fusion, the gp120 fragment is shed. The gp41 ectodomain becomesexposed after shedding gp120. Binding sites for C1q and factor H on gp41become unmasked.

HIV activates human complement systems even in the absence of specificantibodies. (Stoiber, H, J. Ann. Rev. Immunology, Vol. 15, 649-674(1997)) This would result in viral inactivation if complement wereunimpeded. The complement process if unimpeded would produce membraneattack complex (MAC), triggering virolysis. However, HIV avoidsvirolysis by incorporating into its structure various molecules of thehost (e.g., DAF/CD55) that regulate complement. HIV includes thesemolecules in the virus membrane upon budding from infected cells, or byattachment to the gp41 and gp120 structures. (Stoiber, H., J. Ann. Rev.Immunology, Vol. 15, 649-674 (1997)) This interaction with complementcomponents enables HIV to take advantage of complement components toenhance infectivity, follicular localization, and broaden its targetcell range. At the same time, HIV defends against the humoral arm.

Proteins such as Factor H and CR1 have both cofactor and decayaccelerating activities on the C3 convertases. (Stoiber, H, J. Ann. Rev.Immunology, Vol. 15, 649-674 (1997)) C3b integrity is essential for thecomplement cascade to culminate in cell lysis. C3b is rapidly cleaved bya serine protease (complement Factor 1-CF1) after interaction withappropriate complement receptors. Proteins that mediate this reactionpossess cofactor activity for CF1. Some proteins down regulatecomplement activation by inhibiting the assembly and/or by favoring thedissociation of C3b and C4b generating enzymes (convertases). Thisactivity is termed decay acceleration and is characteristic of the CD55(DAF) protein molecule.

Serum lacking Factor H will lyse HIV and infected cells, but not cellsthat are uninfected. (Stoiber, H., J. Exp. Med., 183:307-310 (1996)) Inthe presence of Factor H, lysis of HIV has been shown to occur when thebinding of Factor H was inhibited by a monoclonal antibody directed to aFactor H binding site in gp41. Human serum that is devoid of Factor Heffectively lyses HIV virions. But to date, there has been no indicationof how to implement this growing knowledge of the relationship of HIVand Factor H to the human complement.

Related Art

Despite profound efforts, there is no curative vaccine for HIV. Varioussteps of the HIV life cycle have been targeted by inventors. To date,research has not found a composition that would foster an effectiveimmune response against the immunosuppressive retrovirus HIV-1. Most HIVvaccines use portions of the envelopes of surface glycoproteins (gp160,gp120, and gp41) of the virus in an attempt to induce production ofneutralizing antibodies against the envelope spikes of the virus.(Johnston et al., 2001) Some have been successful in producing hightiters of neutralizing antibodies. The thought behind this approach isthat the antibodies that bind to these glycoproteins would neutralizethe virus and prevent infection. A functioning immune system could thenactivate the complement system, which would cascade to lysis and destroythe virus. However, the impairment of humoral response described abovelimits the effectiveness of these vaccines. A number of drugs orcompositions (AZT, ddl, ddC, d4T and 3TC) inhibit reverse transcription.These 2′,3′-dideoxynucleoside analogs can be effective against certainstrains, but are vulnerable to the genomic mutability of HIV. (Deeks,Steven, The Medical Management of Aids, Ch. 6 (6th ed. 1999)) Thesemedications also face problems of toxicity, cost, complex treatmentregimens, drug-drug interactions, as well as drug resistance.

Interfering with other aspects of the HIV life cycle is less common.Some efforts have targeted interactions between HIV Pr55^(Gag) and thecellular membrane. A few efforts, such as U.S. Pat. No. 6,627,197 toKeener et al., employ the N terminus of the Pr55 protein (which attachesto myristic acid) as a target molecule for protease activated ricin inorder to kill infected cells. However, there remains a need forimmunogenic compositions and methods that employ the amino terminal endof the matrix protein (p17MA) and covalent binding site for myristate onthe HIV virus while stimulating individual elements of both the cellularand humoral immune responses.

SUMMARY OF THE INVENTION

As described above, HIV is capable of infecting both dividing andnon-dividing cells, such as macrophages. Thus, in addition to impairingimmune response by attacking or binding complement regulators, HIVimpairs cellular portions of the immune system. Myristate binds to thematrix protein, triggering association with the cytoplasm of cellmembranes. Accordingly, the present invention is an immunogeniccomposition based on the covalent binding site for myristate on the HIVmatrix protein, including the amino terminal end of the matrix protein(p17MA), and a method for preparing and using the same. The presentinvention contemplates three categories of embodiments: protein orprotein fragments, messenger RNA, or DNA/RNA. DNA/RNA compositions maybe either naked or recombinant. The present invention furthercontemplates use with a variety of immune stimulants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows is a depiction of the human complement cascade pathways.

FIG. 2 depicts the categories of embodiments for amino terminal end ofthe matrix protein (p17MA) and the covalent binding site for myristateon the HIV virus within the present immunogenic composition.

FIG. 3 is a graphic of the exemplary carriers available for recombinantDNA.

FIG. 4 is a chart that demonstrates splicing of genetic materialencoding the genetic material for the matrix protein covalent bindingsite for myristate into recombinant bacterial compositions or vaccines.

FIG. 5 is a chart that demonstrates splicing of genetic materialencoding the genetic material for the matrix protein covalent bindingsite for myristate into recombinant viral compositions or vaccines.

FIG. 6 is a list of immune stimulants for use with naked DNAcompositions

FIG. 7 describes customary routes of administration for DNA.

FIG. 8 is a schematic drawing showing the chain structures of C3 and CVFand their relationship.

DESCRIPTION OF THE INVENTION

A. Introduction

The present invention is an immunogenic composition based on thecovalent binding site for myristate on the HIV matrix protein, includingthe amino terminal end of the matrix protein (p17MA). Both the matureand immature form of the matrix protein may be used. Further, thegenetic sequence encoding the protein may also be used to produce arecombinant embodiment.

In addition to the immune functions described above, another function ofthe immune system is the creation of a “memory” of an antigen. A laterexposure to the same antigen might then prompt a more effective earlyresponse. This memory is created by antigen specific lymphocytes. Thus,memory lymphocytes, along with other cells and factors, provide bothimmediate protection in peripheral tissue and mount recall responses insecondary lymphoid organs. When activated, lymphocytes proliferate,which expands the population of clones of antigen specific lymphocytesas part of the immune response. The new, antigen specific lymphocyteswill be either effector cells or memory cells that are available forresponse in the event of a later exposure. Immune memory enables the useof immunogenic compositions as vaccines.

Also as described above, the matrix protein, along with the Pr55^(gag),control (1) nuclear targeting signal of the matrix protein; and (2) thestrong localizing signal to the cell plasma membrane of Pr55^(gag).(Lee, Young-Min, et al., J. of Virology, pp 9061-9068 (November 1998))The myristic acid moiety attached to the terminal end of the matrixprotein is clearly critical for Gag membrane binding. Mutations ofmyr-MA in HIV-1 can lead to an aberrant accumulation of myr-Gag in thenucleus, and mutations at proximate sites have been shown to reduce Gagmembrane binding. (Tang, Chun, Proc. Nat'l Acad. of Sci. 101: 517-522(January 2004)) In addition, the amino acid sequence of the 11 aminoacids at the N-terminal region of the MA molecule has been sequenced:GARASVLSSGG. Thus, the HIV virus is not able to actively replicateinfectious virions if mutations occur within this 11 amino acidsequence. (Ono, 1999) (Resh, 2004) Therefore this amino acid sequence ishighly conserved, and can be used for subunit vaccines, recombinantvaccines, naked nucleic acid vaccines and mRNA vaccines.

B. Subunit Compositions

The present subunit immunogen is comprised of a peptide or portionsthereof, or the genetic sequences encoding for the protein or proteinsegments in order to create an immune response and immune memory. In thepresent invention, the desired immune response is directed to theGARASVLSSGG peptide, or portions thereof or the encoding genes.Importantly, the composition should be presented properly to the immunesystem. Isolation and use of nucleic acids, peptides, and proteins arefamiliar to those of ordinary skill in the art, and as described herein.

One of the advantages of a subunit composition is a lack of infectivityin therapeutic applications. Therefore subunit compositions may servewhen a virus is extremely virulent, as with HIV. Some viruses such asHIV undergo profound mutation and therefore an attenuated strain used ina vaccine or therapy can undergo spontaneous reversion to a morevirulent strain. Therefore with HIV the use of live viral vectors wouldbe risky. Also subunit compositions or vaccines can be used when thevirus cannot be grown conveniently in culture. Subunit compositions maybe produced quickly and relatively inexpensively.

For example, a subunit vaccine is currently available using thehepatitis B virus surface antigen obtained by expression of a clonedgene in yeast cells. This vaccine has been successfully used in Taiwanand it appears to have reduced the incidence of primary liver cancer inyoung children. (Wagner, 1999)

Direct administration of a protein would not induce a cell-mediatedresponse in the same way that a live virus vaccine would. Yet theadvantages of a subunit vaccine include a lack of potential infectivity,either mild in the case of an attenuated strain or severe in the case ofthe virulent strains. Further, the present invention is contemplated foruse in conjunction with immune stimulants and other immunogeniccompositions.

A strong stimulation of B cells and an antibody response are evidentagainst all of the major HIV proteins soon after infection. (Goudsmit,1988) For unknown reasons, this does not lead to the production ofprotective or effective neutralizing antibodies. On the contrary theseantibodies may enhance uptake of HIV by cells other than CD4lymphocytes, and thereby promote a more efficient localization in theantigen presentation cells (APC), due to deposition of complementfragments on the virus surface. (Stoiber, 1997) In the conversion ofneutralizing antibodies into enhancing antibodies, follicular dendriticcells may play an important role. So far, efforts to generate effectiveneutralizing antibodies by vaccination have been unsuccessful. (Cohen,P. T., et al., The AIDS Knowledge Base, Ch 22 (3rd ed. 1999))

Thus, the composition of the present invention includes a method forinducing an immune response in a human or other animal. Generalreferences to “animal” include humans. The method comprises preparingthe composition and administering to an animal capable of mounting ahumoral or a cellular immune response. An immune response may bedetected using common methods of measurement known in the art. Thepresent invention may be used to develop laboratory tools and researchimmune response. Furthermore this invention will aid the development ofa vaccine for administration to an HIV infected subject or for producingan immune response in a subject that is not infected, but for whom animmune response is desired.

C. Method of Preparation

A variety of methods of development, preparation, and administration arecontemplated by the present invention. It is expected that such methodsshall be selected based on efficacy for the particular strain andresponse of the subject animal. As shown in FIG. 2 this subunitcomposition may be categorized for preparation purposes as protein orpeptide isolation, messenger RNA, or nucleic acid DNA/RNA expression.Thus, matrix protein includes matrix protein peptides or portions, andgenetic expression or material thereof.

Thus, the present invention may be prepared using any one or more of avariety of methods available to those in the field, including but notlimited to:

-   -   1. Purification and isolation of the covalent binding site for        myristate on the HIV matrix protein, including the amino        terminal end of the matrix protein (p17MA);    -   2. Messenger RNA cloning expressing the covalent binding site        for myristate on the HIV matrix protein, including the amino        terminal end of the matrix protein (p17MA); or    -   3. Recombinant DNA/RNA cloning and expression of covalent        binding site for myristate on the HIV matrix protein, including        the amino terminal end of the matrix protein (p17MA) into a        suitable bacteria such as escherichia coli, or yeast, or virus,        or naked DNA/RNA of the covalent binding site for myristate on        the HIV matrix protein, including the amino terminal end of the        matrix protein (p17MA). (Aroeti, 1993)        Antigen presenting cells take up exogenous proteins by        phagocytosis, leading to presentation of the immunogen and        immune response. With reference to the list above, embodiment 1        relies on protein fragments, while embodiments 2-3 rely on        nucleic acids and recombinant technology. Embodiments 2-3 may        also include the synthetic in vitro manufacturing of the nucleic        acid. (Aroeti, 1993)        C.1. Protein Based Compositions

The epitope of the protein may be isolated from a single viral particleor a viral culture. In the case of a single particle, enzymatic(proteolytic) degradation may be used. For example, a mature protein maybe isolated from viral particles by degrading and enzymaticallydigesting the mature viral particles into individual protein components.“Purification” means merely that the protein is sufficiently free ofother cell subunits or contaminants so as to enable therapeutic use. Thecomposition need not be totally pure. The protein portion may also beisolated from a viral culture. Each protein of the viral structure isproduced in quantities exceeding that necessary for viral replication.Therefore, individual viral proteins may be isolated and separated fromviral isolates based on that protein's characteristic size, shape,solvency characteristics, electrostatic potential, density and/orbuoyancy and sedimentation rate in a variety of media. Therefore, thisapproach involves the use of specific protein fragments or peptides toelicit an immune response.

C.2. Nucleic Acid Based Compositions

In general, nucleic acid based compositions may comprise naked DNA/RNA,recombinant DNA/RNA, or messenger RNA. A composition based on naked DNAwould use the DNA of the viral antigen encoding the binding site thathas been stripped of histones (small unfolded chromosomal proteins) orprotein, usually by exposure to detergents or phenols. Recombinant DNAis genetically engineered DNA made by recombining fragments of DNA fromdifferent organisms, as discussed in detail below. DNA/RNA or mRNA forboth embodiments may be isolated, purified, and amplified usingprocedures that are known in the art, and are partially describedherein.

In addition, and as described below, mRNA based immunogenic compositionsand vaccines may be an alternative concept to using naked DNA/DNA orrDNA sequence coding for protein. Messenger RNA is an intermediarybetween the two (DNA and protein), and can be used to transfect cellsand undergo translation within a host cell to produce the viral proteinsin question.

C.2.1. Isolation of DNA and RNA

Procurement of nucleic acid(s) requires three basic steps (1) lysing ofthe cells to expose the nucleic acids preferred for processing; (2)separation of the nucleic acids from other cell components; and (3)recovering of the nucleic acid in purified form. (Nicholls, Desmond, AnIntroduction to Genetic Engineering, Ch. 3 (2d ed. 2002)) “Purification”means merely that the nucleic acid is sufficiently free of other cellsubunits or contaminants so as to enable therapeutic use.

A plethora of modalities may be used to recover nucleic acids. Many arequite simple requiring only a few steps. More complex purificationprocedures involving several different stages are standard in theindustry. Commercially available kits readily enable purification ofnucleic acids from a range of sources.

The first step in any isolation protocol is disrupting the startingmaterial. The method used to open cell walls should be as gentle aspossible, preferably utilizing enzymatic degradation of cell wallmaterial and detergent lysis of cell membranes. If more vigorous methodsof cell disruption are required, there is the danger of sheering largeDNA molecules and this can hamper the production of representativerecombinant molecules during subsequent processing.

Following cell disruption, cell proteins are removed. Phenol orphenol/chloroform mixture is often used in the extraction procedure.Upon centrifugation to separate the phases, protein molecules partitioninto the phenol phase and accumulate at the interface. Due to theirinherent hydrophilicity nucleic acids remain mostly in the upper aqueousspace and may be precipitated from solution using isopropanol orethanol.

If a DNA preparation is required, the enzyme ribonuclease (RNase) can beused to digest the RNA in preparation. If mRNA is needed for the cDNAsynthesis, a further purification can be performed usingoligo(dT)-cellulose to bind to the poly (A) tails of eukaryotic mRNAs.This gives substantial enrichment for mRNA and enables mostcontaminating DNA, rRNA and tRNA to be removed.

Gradient centrifugation is frequently used to isolate DNA, particularlyplasmid (pDNA). DNA is dissolved into a caesium chloride (CsCl) solutionand spun at high speed in an ultracentrifuge. Over time (in some casesup to 48 hours) a density gradient is formed. The pDNA forms an easilyidentifiable band or line at one position in the centrifuge tube. Thisband is devoid of cellular contaminants and may be removed. Usingdialysis, the CsCl is removed to give a pure preparation of pDNA. Sizeexclusion chromatography can be used as an alternative toultracentrifugation. Many plasmid DNAs however, are commerciallyavailable. (Nicholls, 2002)

Amplification of a preferred DNA sequence can be accomplished by thepolymerase chain reaction (PCR). (Nicholls, 2002). Simplicity, eleganceand high specificity characterize PCR, which has replaced traditionalcloning methodology. In the PCR process the DNA duplex is heated,thereby denaturing and unwinding the double helix and separating thestrands. Each single strand is copied by a DNA polymerase. The processis repeated many times resulting in an exponential increase in thenumber of copies.

C.2.2. Recombinant Technologies

The methods used in producing recombinant DNA are conceptuallystraightforward and known in the art. Genes of the HIV matrix proteinmay be engineered into the DNA of a carrier, such as Escherichia coli; alist of suggested carriers is in FIG. 3. As shown in FIG. 4 bacterialcarriers may include rDNA by plasmid, chromosome integration, or acombination. As shown in FIG. 5, viral carriers may support rDNA bychromosome integration, insertion of proteins encoded by the donor DNAinto the viral coat, or a combination. When the carrier reproduces, theimmunogen is propagated if the immunogen is inserted into the hostchromosome. Plasmid DNA can undergo replication within a non replicatingcell. The cutting or isolation of the genes with restriction enzymes isas described herein and known.

Preparation of rDNA

Electrophoresis enables the separation, identification, and purificationof DNA fragments. The porosity of the matrix determines the degree ofseparation achieved. Two gel types are commonly used in the field,agarose and polyacrylamide. Agarose, extracted from seaweed, isavailable commercially as a dry powder, which is melted in buffer at anappropriate concentration. On cooling, agarose sets or gels.Polyacrylamide gels are used to separate small nucleic acid moleculesbecause the pore size of polyacrylamide is smaller than agarose.Polyacrylamide can separate DNA molecules that differ in length by onlyone nucleotide. Electrophoresis may be carried out by placing nucleicacid samples in a gel and applying an electrical potential across it.DNA contains negative charged phosphate groups and will thereforemigrate towards the positive electrode. When a marker dye, usuallybromophenol blue (added to the sample prior to loading), reaches the endof the gel the electrical potential is removed. The nucleic acids in thegel may be visualized by staining with the intercalating dye ethidiumbromide and examined under UV light. (Nicholls, 2002) Large DNAfragments containing as many as 100,000 base pairs can be separated byanother process known as pulsed gel electrophoresis.

Pulsed field gel electrophoresis (PFGE) and simple gel electrophoresispermit DNA fragments to be separated on the basis of size: the smallerthe fragment, the more rapid the migration. Overall rate of migrationand optimal range of size for separation are determined by the chemicalnature of the gel and by the degree of its cross-linking. Highly crossedlinked gels optimize the separation of small DNA fragments. The dyeethidium bromide forms a brightly fluorescent adduct as it binds to DNA.Small amounts of separated DNA fragments can be isolated on gels. Thisdye binds between the DNA bases (intercalates) and fluoresces orangewhen illuminated with ultraviolet light. (Nicholls, 2002) Theidentification of a specific DNA fragment can be accomplished by probescontaining complementary sequences.

All methods of electrophoresis rely on the polyanionic nature of nucleicacids (RNA & DNA, single stranded and double stranded) at neutral pH,i.e., nucleic acids carry multiple negative charges on the phosphategroups. This means that the molecules will migrate towards the positiveelectrode when placed in an electric field. As negative charges aredistributed evenly along the DNA molecule, the charge/mass ratio isconstant, thus mobility depends on fragment length. The technique ispreferably executed on a gel matrix which separates the nucleic acidmolecules according to size. (Nicholls, 2002)

Restriction enzymes or endonucleases allow bacteria to distinguishbetween homologous and heterologous DNA. These enzymes hydrolyze andcleave DNA at specific sites known as restriction sites. Thisspecificity of sequence recognition allows the precise selectivity ofDNA fragment preparation, which is the foundation for DNA vaccines.Bacteria that possess a restriction enzyme system disguise recognitionsites in its own DNA by modifying them. The addition of a methyl groupto an adenine or cytosine residue near or at the site of cleavageprotects its own nucleic acid. (Brooks, Geo., Medical Microbiology 102(23rd ed. 2004))

Restriction modification systems of bacteria fall into two broadclasses: Type 1 systems in which the restriction and modificationactivities are combined in a single multi-subunit protein, and Type 2systems which consist of separate endonucleases and methylases. (Brooks,2004)

An analogy between restriction endonucleases that have become standardlaboratory devices and a surgeon's knife is evident. Restrictionendonucleases are usually named by a three or four letter abbreviationof the named organism from which the enzyme has been isolated. (Brooks,2004) The generic and specific names of the organism in which the enzymeis formed are used to provide the first part of the designation whichcomprise the first letter of the generic name and is the first twoletters of the specific name. Thus an enzyme from the strain ofEscherichia coli is termed Eco and one from Bacillus amyloliquefaciensis Bam. (Nicholls, 2002)

Restriction endonucleases generally cleave phosphodiester bonds in bothDNA strands in a mirror like fashion. A restriction enzyme recognizesand cleaves at the same DNA sequence and only cleaves at that particularsequence. Most of the DNA sequences recognized by restriction enzymesare palindromes; that is, both strands of DNA have the same basicsequence running in opposite directions on either side of the axis ofsymmetry when read in a 5′ to 3′ direction (self-complementary). Thecuts made by these enzymes are usually “sticky” (i.e., the products aresingle-stranded at the ends with one strand overhanging the other.)However, sometimes the products are blunt with double stranded ends.Over five hundred restriction enzymes with different specificities havebeen isolated and characterized. Most are readily available aslaboratory tools.

Restriction fragments of DNA may be used to identify variations in basesequence in a gene. However they can also be used to synthesize arecombinant DNA also called chimeric DNA, which is composed of moleculesof DNA from different sources that have been recombined in vitro. Thesticky ends of two unrelated DNA fragments may be joined to each otherif they have complementary sticky ends. Complementary ends may beobtained by cleaving unrelated DNAs strands with the same restrictionenzyme if the restriction enzyme recognizes palindromic strands. Afterthe sticky ends of the fragments base pair with each other, thefragments can then be covalently attached by the action of a DNA ligase.(Smith, Coleen, Basic Medical Biochemistry: A Clinical Approach, Ch. 17(2d ed. 1996)) DNA ligase is a cellular enzyme that repairs brokenphosphodiester bonds that may occur at random or as a consequence of DNAreplication or recombination. (Nicholls, 2002) The DNA ligase most oftenused is T4 DNA ligase, which may be purified from E. coli cells infectedwith bacteriophage T4. Although the enzyme is most efficient whensealing gaps in fragments that are held together by cohesive ends, itwill also join blunt-ended DNA molecules together under appropriateconditions. DNA ligase produces a phosphodiester bond between a 5′phosphate and a 3′ hydroxyl (OH) group. The enzyme is most effective at37° C., but may be used at lower temperatures. Thermodenaturation of thesingle strand ends however, occurs at higher temperatures (37° C.).Therefore this enzymatic process if often accomplished at lowertemperatures to affect a higher purity although the overall process issomewhat slower. (Nicholls, 2002)

The length of DNA fragments produced by restriction enzymes variestremendously because of the individuality of DNA sequences. Mostrestriction enzymes recognize palindromic sequences which occur somewhatrandomly. Furthermore the average length of a DNA fragment isdetermined, in large part, by the number of specific base pairsrecognized by the restriction enzyme. Restriction enzymes recognizing upto 15 base sequences have been characterized, however most recognizefour, six, or eight base sequences. Recognition of four bases yieldsfragments with an average length of 250 base pairs, and therefore isgenerally useful for analysis or manipulation of gene fragments. As thenumber of base pairs recognized by the restriction enzyme increases theaverage length of the nucleotide sequence increases logarithmically. Forinstance restriction enzymes that recognize six bases produce fragmentswith an average size of about 4,000 base pairs. Restriction enzymes thatrecognize eight bases produce fragments with a typical size of 64,000base pairs and are therefore useful for analysis of larger geneticregions. (Brooks, 2004)

In the production of DNA vaccines, plasmid DNA derived from eukaryoticcells such as bacteria and yeast is often used as the donor vehicle. Aplasmid is a genetic particle physically separate from the nucleus ofthe host cell. The nuclei of prokaryotes are not enveloped. Plasmid canindependently function and replicate, that is independent of the nucleusof the cell. Plasmids usually confer some survival or growth advantageto the host cell, but are not essential to the cell's basic function.For example, a resistance plasmid carries genes responsible forantibiotic or antibacterial drug resistance. Plasmids are small circlesof DNA; however the three dimensional structure is often that of afigure eight or more complex structure. Nonetheless, the small size ofplasmids renders them amenable to genetic manipulation in vitro.Furthermore, after genetic manipulation their small size permitsintroduction into other cells. Therefore, plasmids are frequently usedin genetic engineering and are the basis of most DNA vaccines. (Brooks,2004)

Because many restriction enzymes cleave asymmetrically and produce DNAfragments with cohesive (sticky) ends, hybridization of DNA is possible.This DNA can be used as a donor with plasmid recipients to formgenetically engineered recombinant plasmids. Cleavage of a plasmid withthe same restriction enzyme produces a linear fragment with cohesiveends that are identical to each other. To prevent the two ends of theplasmid from reannealling enzymatic removal of the free phosphate groupsfrom these ends is performed. This ensures that the original circularplasmid is structurally incompetent and cannot function. Ligation in thepresence of other DNA fragments from other sources containing freephosphate groups produces recombinant plasmids or chimeric plasmidswhich contain DNA fragments as inserts in covalently now circular DNA.Plasmids must be in a circular form in order to replicate in thebacterial host. (Brooks, 2004)

The amino acid sequence of the present subunit, the amino terminal endof the matrix protein (p17MA) and the covalent binding site formyristate on the HIV virus, has been deduced. Each amino acid is codedby a separate codon. A codon is a set of three consecutive nucleotidesin a strand of DNA or RNA that provides the genetic information to codefor a specific amino acid which will be incorporated into a proteinchain or serve as a termination signal. Therefore, knowledge of thepresent subunit permits deduction of the nucleotide sequence of the DNAand/or RNA for the amino terminal end of the matrix protein (p17MA) andthe covalent binding site for myristate on the HIV virus. The origin forelongation of a DNA sequence is determined by a DNA primer that can besynthesized by known nucleotide synthesizing devices for chemicaloligonucleotide synthesis. Such devices can produce DNA strandscontaining 75 or more oligonucleotides. (Brooks, 2004)

Chemically synthesized oligonucleotides can serve as primers forpolymerase chain reaction (PCR) which is a procedure that allowsamplification and sequencing of DNA between the primers. Thus, in manyinstances, DNA need not be cloned in order to be sequenced or to be madeavailable for engineering.

DNA sequencing can also be performed using the Maxam-Gilbert technique,which relies on the relative chemical liability of different nucleotidebonds and the Sanger (dideoxytermination) method, which disrupts theelongation of DNA sequences by incorporating dideoxynucleotides into thesequences. Furthermore a procedure known as shotgunning allows thesequencing and analysis of entire genomes in viruses. In this procedure,DNA is randomly fragmented to create a random fragment library. Theseunordered fragments are sequenced by automated DNA sequencers and may bereassembled in correct order using computer software available in thefield. (Brooks, 2004)

The essential components of a plasmid DNA designed for vaccinationinclude a start signal (promoter-enhancer) and stop signal(polyadenylation signal/transcript termination sequence). The start andstop signals can be selected from a variety of sources viral, bacterialor mammalian. A marker of activity of the plasmid such as antibioticresistance or specific enzymatic activity can be included and may beadvantageous if only to demonstrate that a fully functional plasmid hasbeen developed. It is also advantageous to include intron-containingsequences that have been shown to greatly improve expression withintransfected cell lines for many constructs even through introns containsequences that are ultimately not translated into protein. Thepromoters/enhancers that have been mostly used for DNA vaccines are theCMV immediate early promoter (pCMVIE) enhancer and the Rous sarcomavirus (RSV) LTR. Hundreds of plasmids are available commercially fromdifferent suppliers. A basic plasmid vaccine vector is known as V1J.This is comprised of pCMVIE, intron A derived from CMV, bovine growthhormone (BGH) polyadenylation/transcript termination sequence and a gene(amp^(r)) coding for ampicillin resistance. A pUC plasmid DNA sequencefrom which the lac operon and multicloning site have been deleted,serves as the basic construct for this recombinant plasmid structure.Two separate restriction enzyme sites have been mapped for insertion ofdonor DNA. V1J does not replicate in mammalian cells and does notcontain any sequences known to promote plasmid integration into hostgenomic DNA ensuring a wide safety margin. Furthermore it can beproduced in large quantities by growth in E. coli. These properties helpensure the safety of the recombinant DNA process by minimizing theprobability for cell-transforming integration events.

Best results for vaccination in animals have been obtained by usingnormal saline solutions of plasmid. Other vehicles including solutionsof bupivicaine and sucrose have been used, but there has been noenhanced immunogenicity with these methodologies in animals. (Kaufman,Stefen, Concepts in Vaccine Development, ch 3.7.3, (1996)) A smallpercentage of myotubules take up and express DNA following intramuscularinjection of a plasmid saline formulation. This however, has beensufficient for obtaining significant immune responses. (Kaufmann,Stefan, Concepts in Vaccine Development Ch. 3.7 (1996))

Both humoral and cytotoxic T cell responses are noted to occur withnaked DNA vaccines. Strong proliferation of T cells was observed at lowDNA doses in animal models down to one microgram even in the absence ofmeasurable antigen-specific serum antibody responses, indicating thatless antigen may be required to elicit T cell responses by DNA vaccinesthan for antibody generation. Therefore, since the most likely correlateof immunity to HIV disease would be a robust cytotoxic T cell responsedirected toward HIV disease, less (antigen) with HIV vaccine technologymeans more. The development of a strong humoral response to HIV diseasehas been associated with a poorer prognosis. Low dose DNA vaccinesstimulate the production of Type 1 helper T cells (T_(H)1). TH1 cellsgenerate cytokines II-2 and gamma-interferon which have been shown topromote cellular immune responses by stimulating CD8⁺ activity.(Kaufmann, 1996)

For HIV infections, strong T_(H)1-like responses have been important inmaintaining high CD4 cell counts and low viral titers as well asprevention of secondary opportunistic infections. (Kaufmann, 1996)

The advantages of expressing antigens in the host rather thanadministering antigens such as inactivated viruses, recombinant proteinsor peptides, include the following: (1) circumventing potential loss ofantigenicity by an inactivation process (e.g., chemical cross linking)inherent in the host cell; (2) synthesis of proteins with conformationand post translational modifications including carbohydrate and lipidlinkages encoded by the host cell; (3) intracellular antigen processingand presentation by MHC class I molecules leading to the induction ofcytotoxic T lymphocyte (CTL) responses; and (4) allowing for MHCdeterminant selection. (Kiyono, Hiroshi, Mucosal Vaccines Ch. 8 (1996))

Antigen presentation after IM DNA vaccination results in a robustcytotoxic T cell response. Three models for inducing the CTL responsewith IM DNA vaccines have been proposed:

-   -   1. Uptake of DNA and expression of antigens by antigen        presenting cells including dendritic cells, macrophages and        langerhans cells;    -   2. Antigen presentation by transfected myocytes acting as or        assuming the role of antigen presenting cells; and    -   3. Transfer of antigens from transfected myocytes to antigen        presenting cells which in turn present the antigen to the        appropriate T cell. (Kiyono, 1996)

DNA vaccines have been used to elicit specific immune responses,antibody, CD8 cell and CD4 cell, against a variety of antigens in animalspecies, including but not limited to the following:

-   -   1. Hepatitis B surface antigen in mice (Davis, et. al., 1993,        1994)    -   2. Herpes simplex virus 1 glycoprotein B in mice (Manickan et.        al., 1995)    -   3. Bovine herpesvirus 1 glycoprotein IV in cattle (Cox et. al.,        1993)    -   4. Rabies virus glycoprotein in mice (Xiang, et. al., 1994,        1995)    -   5. Malaria circumsporozoite protein in mice (Sedegah, et. al.,        1994; Hoffman et. al., 1994)    -   6. Leishmania gp63 in mice (Xu and Liew 1995)    -   7. Lymphocytic choriomeningitis virus (LCMV) NP in mice (Pedroz        Martins, et al. 1995; Yokoyama et. al., 1995)    -   8. Carcinoembryonic antigen in mice (Conry, et. al., 1994)    -   9. MHC class I antigen in rats (Geissler, et. al., 1994)    -   10. Cottontail rabbit papillomavirus (CRPV) L1 in rabbits        (Donnelly et. al., 1996)    -   11. M tuberculosis antigen 85 complex proteins in mice (Huygen        et. al., 1996) (Kaufmann, 1996)

More specifically, the ability of DNA vaccines to induce CTL responseshas also been demonstrated several times. It was first demonstratedusing influenza NP (nucleoprotein). NP is a conserved internal proteinof the virus and a target for cross reactive CTL. The NP DNA induced aCTL response in mice which demonstrated an element of longevity implyingthe potential for vaccination. Interestingly cell mediated immunityinduced by DNA encoding influenza NP or matrix protein also played arole in protection of ferrets as measured by reduction of virus sheddingin nasal secretions. DNA vaccine induced CTL response has beendemonstrated for the following as well:

-   -   1. Rabies virus glycoprotein (Xiang, et al., 1994)    -   2. Malaria circumsporozoite protein (Sedegah, et al., 1994)    -   3. Lymphocytic choriomeningitis virus NP (Pedroz Martins, et        al., 1995; Yokoyama, et. al., 1995; Zarozinski et al., 1995)    -   4. HIV envelope protein (Wang, et al., 1994; Shiver et al.,        1995)    -   5. Human Factor IX (Katsumi, et al., 1994)    -   6. MHC class I (Geissler, et al., 1994; Plautz, et al., 1994;        Hui et al., 1994)

Detection of CTL responses for one to two years after immunization hasbeen noted in some of the above models. Dosing of the DNA vaccine shouldstart at 1 mcg. CTL assays should be performed and the lowest dose atwhich an adequate CTL response is noted is a preferable dose foradministration.

As discussed below, cationic lipids formulated with IM DNA vaccineactually resulted in a lower level of gene expression. However, the useof cationic lipids to facilitate DNA uptake has been noted with mucosaldelivery systems. Cationic lipids facilitate DNA uptake on mucosalsurfaces via a non-specific mechanism or a specific plasma membranetransport mechanism yet to be characterized. Mucosal delivery of DNA canpotentially transfect many cell types lining the GI and GU tract as wellas the cells beneath their respective basement membranes includingPeyer's patches which are preferred sites of HIV replication. Inaddition to potential facilitation of cellular uptake on mucosalsurfaces, cationic lipids also protect DNA from degradation. In vitrostudies have shown that DNA/cationic lipids have a longer half life thanuncomplexed DNA. (Puyal, et al., 1995) Therefore the preferredembodiment for mucosal DNA vaccines will include cationic lipids.

Parenteral administration of DNA vaccines induces strong systemichumoral and cell mediated immune responses (dose dependent), but doesnot result in the generation of significant mucosal immune responses.Therefore in certain instances it may be desirable to design a vaccinethat could induce both mucosal and systemic immune responses. (Kiyono,1996) This can be achieved by DNA vaccines delivered by different routes(parenteral and mucosal). This approach has been tested in severalsystems using parenteral priming followed by mucosal boosting (Keren, etal., Infect. Immun., 56: 910-915 (1988)) and vice versa (Forrest, etal., Infect. Immun. 60: 465-471 (1992)). With some vectors mucosaladministration of DNA/cationic lipids resulted in both local andsystemic immune responses. A recombinant BCG vaccine induced local IgAand serum IgG antibodies against heterologous antigen (Langerman, etal., 1994) and a recombinant Salmonella vector given orally induced cellmediated immunity (Aggarwal, et al., 1990).

A preferred embodiment utilizing DNA vaccine technology would be acombination of a naked DNA vaccine administered parenterally (preferablyintramuscularly) and a cationic lipid/DNA vaccine applied mucosally.

Therefore in summary, to produce a recombinant bacteria DNA vaccine, thefollowing steps will be followed:

-   -   1. Selecting a suitable plasmid vector from commercially        available sources    -   2. Isolating the subject HIV DNA    -   3. Effecting restriction enzyme cleavage/modification of plasmid        DNA and HIV DNA    -   4. Isolating the specified gene(s) from HIV    -   5. PCR amplifying selected HIV DNA gene(s)    -   6. Enzymatically removing free phosphate (PO₄) groups from        plasmid DNA    -   7. Transforming the plasmid DNA into a bacterial cell such as E.        coli.    -   8. Administering ligase to seal the DNA strands together

To accomplish the process of transformation the recipient cells need tobe made competent. Competence relates to the ability of a cell toassimilate foreign RNA or DNA. The steps to accomplish this are:

-   -   1. Soaking the recipient cells in an ice cold solution of        calcium chloride (this induces competency in a way that is still        not fully understood);    -   2. Mixing the plasmid DNA with the cells and incubating them on        ice for 20 to 30 minutes;    -   3. Heat shocking (two minutes at 42° C.) to enable the DNA to        enter the cells;    -   4. Incubating the transformed cells in a nutrient broth at        37° C. for 60 to 90 minutes. This allows the plasmid to become        established and ultimately permit phenotypic expression of the        plasmid nucleic acid; and    -   5. Placing the cells with the plasmid vector onto a selected        media suitable for replication.        As shown in FIG. 3, rDNA/RNA may be delivered by a bacterial or        viral carrier.        C.2.3 Recombinant Carriers        C.2.3.1 Bacterial Carriers

Live attenuated bacteria may serve as carriers for DNA/RNA. Bacteria maycarry and express genes that are encoded with the amino terminal end ofthe matrix protein (p17MA) and the covalent binding site for myristateon the HIV virus on the matrix protein or portions thereof. The bacteriaprovide an environment in which the capsid protein DNA/RNA may beamplified, purified and administered. Bacterial carriers may includethose customary in the art, exemplary types being Salmonella, BCG, E.Coli, Streptococcus gordonii, Lactococci/Lactobacilli, Vibrio Cholerae,Yersinia enterocolitica, Shigella flexneri, and Listeria monocytogenes.Salmonella, BCG, and E. coli are preferable.

Among the bacteria thus far explored for recombination, attenuatedSalmonella sp. has received the most intense scrutiny. Other bacteriaincluding Bacillus Calmette-Guerin (BCG) have also been investigated.Attenuated enteric pathogens including E. coli, Vibrio, Yersinia andShigella have been used as platforms for recombinant vaccine technology.Other organisms generally considered as commensals including the grampositives Streptococcus gordonii, Staphylococcus xylosus and thelactococci or lactobacilli have been used in recombinant methodologies.Recently Listeria monocytogenes has been introduced as a potentialrecombinant vaccine vector. Most of these organisms by virtue of theirability to colonize and/or infect mucosal surfaces lend themselves todelivery to these surfaces. Therefore the gut associated lymphoid tissue(GALT) is being stimulated directly through mucosal immunization ratherthan antibody diffusion from the serum subsequent to parenteralinoculation. GALT including Peyer's Patches is the primary site of HIVinfection and replication in sexual transmission of the disease.

The preponderance of attention is focused on enteric pathogens,particularly Salmonella. The bacteria undergo the process of attenuationbefore recombination can occur. In doing so, the bacteria becomeavirulent and are unable to cause typhoid fever or other salmonelladerived diseases. The first description of such mutation appeared in1951 in the metabolic pathway for p-aminobenzoic acid (pab).Subsequently gal E mutants of S. typhimurium and S. typhi (strain Ty21a)were isolated which resulted in the cytoplasmic accumulation ofgalactose-1-phosphate leading to the lysis of cells. Hoiseth and Stockerin S. typhimurium introduced the widely used salmonella auxotrophicmutant, aro A, which encodes the enzyme5-enolpyruvyl-shikimate-3-phosphate synthetase, an essential element inthe aromatic pathway. Additional mutations made in this pathwayinvolving aro C and aro D genes in S. typhimurium result in highlyattenuated organisms. Mutations in the regulatory genes cya, crp whichencode for adenylate cyclase and the cyclic AMP receptor proteinrespectively have also been proven highly successful. Furthermore thecya and the crp mutations are often used in conjunction with mutationsin asd encoding aspartate gamma-semialdehyde dehydrogenase which isessential for peptidoglycan synthesis. In addition, mutations of otherregulatory genes such as phop (phosphatase) and ompR (outer membraneproteins) have proved successful as attenuators of vaccine vectorstrains. (Hughes, Huw, Bacterial Vectors for Vaccine Delivery, DesignerVaccines Principles for Successful Prophylaxis, Ch. 8 (1998))

Three separate methods have been used for expression of heterologousantigens in salmonella have been delineated: (1) plasmids; (2)integration of the foreign gene into the salmonella chromosome; and (3)transportation of foreign antigens to the cell surface by variouscarrier proteins of the salmonella bacteria including flagellin,Neisseria, IgA protease precursor, 1 anB, phoE, ompA. Other carriers ofepitopes which target alternative cellular compartments include fusionswith maltose-binding proteins (malE), LTB, the C fragment of tetanustoxin (tetC),. -galactosidase, pagC and the core antigen (HBcAg) ofhepatitis B. (Hughes, 1998)

Recombinant salmonella has been used successfully to express a number ofviral antigens with induction of both humoral and cell mediatedresponses to the heterologous antigen in animal studies. Variousproteins of influenza have been successfully expressed using thesalmonella bacterial vector in animals, including the nucleoprotein (NP)and an epitope of the hemagglutinin protein (HA). Other viral DNAsequences have been successfully integrated into salmonella includeshepatitis B virus, HIV, and herpes simplex.

Most studies have used the oral delivery system for foreign antigens butothers have used parenteral immunization protocols. Both can be usedconcomitantly or sequentially with recombinant vaccines. Other variablesthat need to be addressed with recombinant bacterial vaccines with HIVdisease include the targeting of foreign antigens to the specific cellcompartments. Interestingly, BCG and Listeria appear to be moreadvantageous for eliciting a cellular response and therefore would bethe preferable routes for recombinant vaccine technology with HIVdisease. (Hughes, 1998)

Using attenuated salmonella bacteria does have an advantage in that itinitially replicates in the large intestines and immune response occursin Peyer's patches, which are the immunologic vehicles lining theterminal colon and are the sites for initial HIV replication in mostcases where the virus is transmitted sexually. Therefore salmonellabacteria would offer a preferred methodology for recombinant vaccinetechnology with HIV disease.

The techniques of transformation and transfection represent the simplestmethods available for getting recombinant DNA into cells. In the contextof cloning E. coli cells, transformation refers to the uptake of plasmidDNA and transfection to the uptake of bacteriophage DNA. A bacteriophageis a virus that infects bacteria. Like other viruses they contain either(but never both) RNA or DNA, and vary in structure from the seeminglysimple filamentous bacterial virus to a relatively complex form withcontractile tails. Their relationships to the host bacteria are highlyspecific. Transformation is also used more generally to describe uptakeof any DNA by any cell. (Nicholls, 2003)

Only a small percentage of competent cells undergo transformation. Thus,the process can become the rate limiting step in a cloning experimentwhere a large number of individual recombinants is required or when thestarting material is limited. Properly performed, 10⁹ transformed cells(transformants) per microgram of input DNA can be realized, althoughtransformation frequencies of about 10⁶ or 10⁷ transformants permicrogram are more realistic. (Nicholls, 2003)

An alternative to transformation procedures is to introduce DNA into thecell by a physical method. One exemplary technique is microinjection, orusing a very fine needle and injecting the DNA directly into thenucleus. This technique has been used successfully with both plant andanimal cells. The cell is held on a glass tube by mild suction, and theneedle is used to pierce the membrane. The technique requires amechanical micromanipulator and a microscope and is done by hand.(Nicholls, 2003) Microinjection offers a preferred embodiment for DNAbacterial recombinant vaccine production with HIV disease.

C.2.3.2 Viral Carriers

Recombinant viral vaccines may be engineered to express genes from thepathogen against which the host is to be protected. The vector serves asa vehicle to carry the foreign gene into the host, and aftertranscription and translation of the nucleic acid present the proteinencoded by the nucleic acid to the immune system of the host. As withany vaccine, of course, the major criteria for acceptability are safetyand efficacy. Safety may be approached from two perspectives. The safetyof the immunogen can be assured by using viral vectors with good safetyrecords due to prior attenuation or prior vaccination of the host to thecarrier virus. Secondly, viruses may be engineered to improve safety ina rational and reliable manner. (Hughes, 1998) The utilization of viralvectors to which the host has already been immunized does have adisadvantage in that the immunogen would be rapidly destroyed by amemory immune response. Nonetheless some transcription and translationof recombinant DNA or RNA would occur. A preferred methodology would beuse of an attenuated nonvirulent virus (without prior immunization tothe carrier virus) as a carrier for the recombinant vaccine.

Thus, viruses like bacteria or yeast may also be used in recombinanttechnology. As carriers, viruses easily infect cells and stimulatecytotoxic T cell immune responses. Because the carrier virus may be ableto replicate, a full and complete immune response may be generated. Boththe humoral and cellular arms of the immune system would then beactivated. General viral carriers may include Poliovirus, AdenovirusStrains 2, 4, 5, and 7, and Poxviruses. Some of the poxviruses used inrecombinant technology include vaccinia, canarypox, ALVAC (derived fromcanarypox), fowlpox, pigeonpox, and swinepox. Other viral vectors usedin recombinant technology include herpesvirus (HSV-1, VZV (herpeszoster), EBV (Epstein Barr Virus)), Alphaviruses, Paramyxoviruses,Influenza, and Hepatitis D. Of these, a preferred embodiment is based onpoliovirus due to extensive existing knowledge of the virus structureand lifecycle. Prior immunization to Polio would be a consideration inlimiting the immune response. Chronic viral infections such as HSV-1offer an attractive alternative since the host immune system wouldreceive low dose background immunogen stimulating cytotoxic activity.

The introduction of genes from one microorganism into the genome ofanother microorganism may result in a virulent strain. To avoid this,the carrier virus should be modified to ensure that any use of thecomposition in treatment is, in fact, avirulent. This would allow for amyriad of viral mosaic combinations to be developed. The gene(s)introduced may replace genes not required for replication of the carriervirus when it is used as a vaccine or it could be added to the viralgenome. (Wagner, 1999) Methods for practicing recombinant technology inthe production and use of immunogenic compositions or vaccines for viralinfections are known and currently available to those in the field.(Porter, 1995) (Stahl, 1997)

Among the viruses used for recombinant viral vaccine vectors are poxviruses (vaccinia virus which includes fowlpox, canarypox, pigeonpox,myxoma and swinepox), adenovirus (particularly types 2 and 5 which havebeen sequenced and adenovirus types 4 and 7 have been widely used asvaccines, not commercially but for the U. S. military without evidenceof adverse effects), herpes virus, polio virus and influenza virus. HIVgenes have been spliced into vaccinia virus vectors with some limitedsuccess in animals. With adenovirus, genes can be inserted into thenon-essential E3 region (up to four kb) or in the essential E1 region.Interestingly, construction of recombinant adenovirus expressing theglycoprotein B of herpes simplex virus (HSV) from the E3 region has beenperformed by McDermott et al. Inoculating mice with this recombinantvirus produced antibodies specific for gB which neutralized HSV invitro. In addition, mice were protected from a lethal HSV challengeafter a single inoculation with the adenovirus recombinant. Jacobs, etal. have utilized the E1 region to express and non structural gene, NS1,from the tick-borne encephalitis (TBE) virus. They have demonstratedprotection against lethal challenge in a murine model using thisreplication defective system. The E1 deleted adenoviruses have an extrasafety factor introduced by their replication defective nature. The E3gene confers immunoprotection to the virus. Therefore, recombinantadenovirus vectors missing the E3 gene are attenuated and avirulent andrepresent a preferred embodiment using adenoviral vectors withrecombinant viral technology. The gp19 protein encoded by the E3 regionreduces expression of the major histocompatibility complex (MHC) class Iantigens in infected cells. The gp19 protein may act at the level oftranscription, translation, protein modification in the endoplasmicreticulum or Golgi apparatus or combination thereof. Adenovirus vectorsdeficient in this gene may be more efficient in presenting the proteinsencoded in their foreign genes to the immune system in a more effectivemanner eliciting a more robust CD8 cytotoxic response. Furthermore,hepatitis B surface antigen has been expressed from adenovirus strains 4and 7, both with and without deletions of E3, and in animal models agood antibody response was noted in those vectors lacking the E3sequences. Vectors containing a functional E3 sequence generated onlyweak or negligible responses. (Hughes, 1998)

Herpes viruses have a large genome and several genes have beenidentified as non essential in vitro and more importantly in vivo. Thedeletion of non-essential genes would allow recombination at severalsites and allow more than one recombination event per virion. A limitednumber of examples of herpes virus vaccine vectors have been tested in anatural host with some success. For example, Dan Ziji, et al. hasreported the protection of pigs against pseudo-rabies virus as well ashog cholera virus.

Influenza has been recently added to the list of potential viral vaccinevectors in recombinant vaccine technology. Influenza in an uncompromisedhost is relatively nonvirulent. Manipulation of the influenza nucleicacid can be accomplished with reverse genetics. Castrucci, et al. haveconstructed a recombinant influenza virus expressing a CTL epitope fromthe LCMV nucleoprotein in the stalk of the influenza neuraminidaseenzyme which cleaves sialic acid. A single dose of this recombinantvaccine protected mice against future challenge by virulent nonattenuated LCMV. Many influenza strains have been characterized, andmany of those vary only in the hemagglutinin and neuraminidase proteinsthey express. Therefore, different influenza strains can be usedsequentially to vaccinate a host to a specific viral protein without theproblem of developing immunity to the viral vector itself which wouldlimit the effectiveness of repeated inoculations. Cold-adaptedattenuated influenza viruses have been used extensively for years asvaccines. Stocks of these vaccines could be used for recombinant virusvaccines, particularly if several inoculations were required.

Rodriguez, et al. tested the efficacy of recombinant influenza vectors.The CD8⁺ T cell epitope of the circumsporozoite protein of Plasmodiumyoelii, a rodent malaria parasite, was expressed in two distinctinfluenza proteins, hemagglutinin and neuraminidase in the same virion.In addition a vaccinia virus recombinant expressing only one copy of thesame epitope was constructed. Both vectors systems were found to inducecomparative levels of epitope-specific T cells. The most efficaciousprotocol consisted of priming with the influenza recombinant followed byboosting with a vaccinia recombinant. (Hughes, 1998)

Two separate recombinant viral vectors may be used sequentially orconcomitantly for optimum immune response with HIV disease.

Live vaccines against polio (Sabin) are attenuated strains of the virusitself. Although these vaccines proved to be extremely safe andeffective (introduced in 1961), occasional reversion to virulencecomplicated the methodology. The American Academy of Pediatrics endorsedthe older Salk vaccine (introduced in 1955), which is not capable ofactive replication. However, despite its safety, the Salk vaccineproduces a less competent immunologic response. Due to the tightcompartmentalization of the poliovirus virion, only small DNA sequencescoding for a few amino acids can be cleaved into the viral genome forrecombinant technology.

Polio virus is classified as an enterovirus because of its fecal/oraltransmission route. Polio is a plus stranded RNA virus as is HIVdisease. To differentiate between the two, although both are positivesense RNA, the retroviruses require RNA to be converted to DNA by avirion-associated enzyme (reverse transcriptase). Polio however does notrequire a reverse transcription enzyme. The polio RNA functions like acellular messenger RNA. Both viruses are encased in icosahedralstructures. Polio is non-enveloped; HIV however is an enveloped virus.

Polio-specific cellular immune responses have recently been studied. Thegeneration of a cell mediated response to the polio virus has beendemonstrated in orally vaccinated volunteers. (Simmons, et al., 1993;Graham et al., 1993) This is important because as mentioned before, Tcell immunity will be the best correlate with immune protection to HIVdisease. (Kiyono, 1996)

Interestingly, the polio virus can be delivered not only orally butnasally to stimulate both the systemic and mucosal antibodies. Thedevelopment of a recombinant vaccine vector based on polio virus hasbeen facilitated because of the immense knowledge available about thevirus. The complete viral RNA genome has been sequenced and the viralproteins identified. (Kitamura, 1981) (Racaniello, 1981) An infectiouscDNA of the viral genome has been generated, making it possible tomanipulate the virus genetically. (Racaniello, 1981) (Semler, 1984) Thethree dimensional structure of the complete virus is known and the majorantigenic epitopes have been identified on the molecular level. (Hogle,1985) The receptor (PVR) that polio virus utilizes to gain entry intothe cells has been cloned and the nucleic acid sequence has beendetermined. (Mendelsohn, 1989; Ren, 1992) Furthermore, transgenic micehave been bred with expressed polio virus receptor and are thereforesusceptible to polio virus infection. Therefore, an animal model existsto study recombinant polio virus vectors with all diseases, especiallyHIV disease.

The vast information available on the polio virus makes it an idealtarget for the development of recombinant poliovirus/HIV vectors. Sincepoliovirus vaccines can be administered to mucosal sites and since polioreplicates in Peyer's patches after initially inoculating tonsillartissue, recombinant polio vaccines are a preferred embodiment forrecombinant viral vaccines for HIV disease.

The availability of an infectious polio virus cDNA has prompted furtherinvestigation into the regions of the polio virus genome that can bedeleted without compromising the replication capacity of the RNA.(Racaniello, 1981) (Semler, 1984) These RNA molecules or repliconsretain the property for self-replication when introduced into cells.Early studies by Kaplan and Racaniello describe polio virus repliconswith deletions encompassing the majority of the P1 region. (Kaplan,1988) Polio virus replicons containing fragments of up to 1.5 kb of theHIV-1 gag, pol or env genes have been the subject of laboratoryinvestigations. (Choi, 1991) The foreign genes were inserted so thetranslational reading frame was maintained between the remaining capsidsequences encoding the P2- and the P3-proteins. Transfection of theseRNAs into cells resulted in the replication of these genomes as well asthe expression of foreign proteins as a fusion protein with the flankingcapsid proteins. (Kiyono, 1996)

The polio virus cDNA has been modified to accommodate larger genes forexpression of recombinant proteins. In these vectors the complete P1region of the polio virus was deleted, and a replicon was constructedwhich contained the complete gene for HIV-1 gag (approximately 1.5 kb).Transfection of this replicon into cells resulted in the production ofthe HIV-1 Gag precursor protein, Pr55^(gag) which was eluted from thesupernatant of the cells after centrifugation and visualized withelectron microscopy. (Porter, 1996) (Kiyono, 1996)

In conclusion, it is possible to express a wide variety of foreign genesincluding genes encoding glycosylated proteins using the polio virusreplicon system. (Kiyono, 1996)

C.2.4 mRNA Expression

The activation of a host cell results in HIV transcription of viral DNAinto messenger RNA (mRNA). In HIV, viral RNA acts as both a messengerand genomic RNA. The viral DNA is transcribed into mRNA. The viral mRNAmigrates into the cytoplasm where it becomes associated with cellularribosomes and cellular transfer RNA to produce viral protein. MessengerRNA is a stable strand of genetic material that communicates the geneticinformation of the virus. Messenger RNA is attractive for use in animmunogenic composition for its stability and efficiency. Messenger RNAis more efficient than DNA in coding for protein.

RNA or DNA encodes for various proteins. An intermediate step is theproduction of mRNA. The mRNA for a protein or group of proteins isidentical to the DNA strand (or RNA strand) encoding for it, with theexception that thymidine in DNA is substituted for uracil in RNA. Alsoin DNA the sugar moiety is deoxyribose in RNA the sugar moiety isribose. The mRNA undergoes the process of capping where at the 5′ end a7-methylguanosine triphosphate is added and at the 3′ end a poly(A)tailof about 100 bases is added to the untranslated segment of the 3′ end.The cap is necessary for the proper binding of the ribosome and the tailsignals an end to the ribosomal translation. Transcription is theprocess where DNA “transcribes” into mRNA. Translation is the processwhere mRNA is “translated” into proteins.

There are many theoretical advantages to mRNA within an immunogeniccomposition. These include but are not limited to: (1) mRNA does notneed to cross through the nuclear membrane; (2) mRNA does not need toenter nucleoplasm; (3) mRNA does not need to integrate into host DNA;(4) mRNA does not need to undergo the process of transcription; (5) thehost translational enzymes and ribosomes are available to the mRNAwithin the cell cytoplasm to allow for translation of the mRNA intoprotein; (6) a quicker immune response should be noted with mRNA incomparison to intracellular DNA because many steps in the production ofviral protein are circumvented; (7) mRNA can be re-used several times sothat many protein sequences can be translated from one mRNA template;therefore only minute quantities of mRNA need enter into the cellcytoplasm; and (8) because the intracellular production of proteins willbe accomplished with mRNA, these proteins will be associated with MHCclass I proteins on the cell surface and will elicit a CD8⁺ cytotoxic Tcell response.

The production of mRNA is straightforward. With the knowledge of aspecific amino acid sequence of a specified HIV protein the RNA sequencecomplementary to this can be deduced. Then the RNA sequence can becapped and tailed at the 5′ and 3′ ends respectively. Furthermore mRNAcan be produced by automated nucleic acid sequencing synthesis, as isknown in the art.

C.2.5 Enhancing CD8+ T Cell Response for Naked DNA/RNA basedCompositions

DNA-based compositions may offer a number of potential advantages overconventional vaccines. Single dosing, long-lasting immunity,cell-mediated immunity as well as humoral responses can be realized withintracellular production of viral particles introduced by recombinantDNA technology. In contrast subunit vaccines based on proteinsinternalized by endocytosis generally do not sensitize cells for CD8⁺ Tcell recognition.

One evasion strategy of HIV and other viral pathogens is to penetrateand replicate in non immunologic cells For example, epithelial cells areinvaded by Chlamydia sp. and Rickettsia sp., while hepatocytes aretargets for Plasmodium sp. and L. monocytogenes. As described above,although HIV targets primarily CD4 cells, other non immunologic tissuesare invaded, such as the central nervous system. In stimulating anenhanced CD8 cytotoxic response, a broader scope of target cells may berecognized by the immune system. As described above, CD8⁺ T cellsrecognize antigens in the context of MHC class I molecules that arepresent on all nucleated cells and enables the CD8⁺ T cells to detectinfected host cells of any type. In contrast, CD4⁺ T cells arerestricted to MHC class 2 expressing host cells and are thus much morelimited in scope. Macrophages, dendritic cells and B cells bear MHCclass I as well as MHC class II molecules. Furthermore, Langerhans cellsof the skin possess both class I and class II MHC proteins. (Kaufmann,1996) Accordingly, constituents enhancing CD8+ T cell response arecontemplated for the present invention. As shown in FIG. 6, a variety ofconstituents may be combined to naked DNA/RNA embodiments to enhanceCD8+ T cell response, some of which are described here.

For example, it has been demonstrated that specific hypomethylated CpGmotifs within bacterially derived DNA can exhibit a potent adjuvanteffect that is, in part, responsible for induction of Th1-type responsethat is a characteristic feature of DNA based vaccines. A significantfeature of DNA based vaccines, unlike most conventional vaccines, is theunique ability to stimulate humoral and cell mediated responses inimmunized animals. The ability to induce a potent Th1-type immuneresponse is of considerable importance because with many pathogens(viral, bacterial, and parasitic), cell-mediated immunity and not thepresence of antibodies is correlated with protection. (Lewis, 1999)

An additional method of enhancing cytotoxic T cell activity is to linkthe mycobacterium tuberculosis heat shock protein 70 (HSP70) to actualnaked DNA/RNA that encodes the subunit. HSP70 is a cytosolic HSP thatfunctions in protein folding, transfer, and degradation. (Chen, 2000)HSP reactive T cells can exert a strong helper effect by reacting toconjugated peptides; HSP can induce a T-helper pro-inflammatory responseand induce the secretion of TNF-α and IFN. (Chen, 2000) Immunologically,calreticulin (CRT), a Ca²⁺ binding protein located in the endoplasmicreticulum, is related to HSPs It associates with peptides delivered tothe endoplasmic reticulum by transporters associated with antigenprocessing and presentation. (Wen-fang Cheng, 2002) CRT enhances CD8activity.

Proteasomal degradation of antigen can enhance MHC class I presentation.(Chien-fu-hung, 2003) Thus, an additional method of enhancing cytotoxicT cell activity is to link gamma-tubulin to the DNA/RNA sequence. Acentrosome is a sub-cellular compartment rich in proteasomes.Centrosomes are important in mitosis and the production of tubules.Centrosomes are also an important locus for MHC Class I antigenprocessing. Linking gamma-tubulin to DNA/RNA will result in cellularlocalization of the protein to the centrosomes, enhancing CD8+ T cellimmune response. (Chan, 2000) Similarly, the present composition may usea DNA/RNA sequence encoding for the lysosome associated membrane protein(LAMP-1) linked to a DNA/RNA sequence for the matrix protein to enhanceB-Cell response. (Chen, 2000) (Chien-fu-hung, 2003)

C.2.6 Enhancing CD8+ T Cell Response for Subunit Based Compositions

As noted above, subunit protein vaccines may not sensitize cells forCD8⁺ T cell recognition. However priming of CTL responses with intactproteins has been achieved by incorporation of the antigen intoimmunostimulating complexes such as ISCOMs (a matrix of lipid micellescontaining viral proteins that deliver antigens to the cytosol andallows induction of cytotoxic T cells) or liposomes. Furthermorecationic lipids have been used to enhance class I MHC pathways ofantigen presenting cells in animals. One cationic lipid used is DOTAP(N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methyl sulfate)which is a commercially available cationic lipid used for DNAtransfection. Other cationic lipids which can sensitize target cells areavailable commercially. These lipids are similar in structure to DOTAPwith two long hydrophobic alkyl chains coupled to one or more positivelycharged ammonium groups. The proposed mechanism of action for thecationic lipids involves an interaction between the macromolecule-lipidcomplex carrying an overall positive charge and the negatively chargedcell surface followed by fusion with the cell membrane. In contrast, pHsensitive liposomes are thought to destabilize upon contact with theacidic environment of the endosome and rupture and/or fuse with theendosomal membrane to release their contents into the cytoplasm.(Walker, 1992)

ISCOMs contain Saponin which is a complex glycoside found in plants.Saponin possesses an adjuvant quality. Saponin has a hydrophilicoligosaccharide sequence of about 8 to 10 monosaccharides. Thepreparation of ISCOMs is know to those familiar with the art. SinceISCOMs also possess a steroid or triterpene their basic structure isamphiphatic. This allows ISCOMs to form a lipid matrix associated withhydrophobic proteins. The lipid quality of ISCOMs allows membrane fusionwith a target cell. The proteins suspended in lipid matrix of the ISCOMsbecome internalized in the target cell and are subjected to immunologicclearance. (Kiyono, 1996)

Formation of complexes between the soluble protein of a subunit vaccineand DOTAP occurs by ionic interactions between the negative charge ofthe protein and the cationic lipid. Thus the maturation or modificationof a subunit vaccine is not required. Association therefore requiresonly mixing of the subunit protein in the DOTAP solution or othercationic lipid prior to application to cells or injection intoexperimental animals or humans. Thus cationic lipids are readilyavailable delivery vehicles for study of intracellular events that leadto class I MHC presentation of antigen and they can serve as analternative to recombinant viruses for enhancing CD8⁺ T cell response toviruses. (Walker, 1992)

The ISCOMs or lipid carriers act as adjuvants but with minimal toxicity.They load proteins and peptides into the cell cytoplasm allowing class Irestricted T cell responses to peptides. Therefore they can be used withsubunit vaccines to enhance CD8 activity. To gain access to thecytoplasm of the cell, the lipid micelles of the ISCOMs fuse with thecell membranes as noted above, and the particles trapped within theISCOMs can be transported to the endoplasmic reticulum. Once inside theendoplasmic reticulum, these particles are bound to newly synthesizedMHC class I molecules. For final protein modification the particles passthrough the Golgi apparatus. They are then transported to the cellsurface as peptide MHC class I complexes. (Parham, Peter, The ImmuneSystem, Ch. 12 (2004))

Therefore, the present composition should preferably be incorporatedinto ISCOMs, liposomes, and/or dissolved in cationic lipids to enhance Tcell activity or to prime the CTL responses

C.3. Conclusion—Method of Preparation

Thus, the present invention comprises both a protein based compositionand a nucleic acid based composition that could be used to induce animmune response against the amino terminal end of the matrix protein(p17MA) and the covalent binding site for myristate on the HIV virus,and to create immune memory thereto. Nucleic acid based compositions maybe DNA, RNA, or mRNA. Recombinant nucleic acid carriers may be bacterialor viral. Preferably, the composition includes one or more constituentsfor enhancing CD8+ T cell response.

Protein based compositions may be developed and administered usingmethods that are known in the art. For the purposes of compositions orvaccines that are based on nucleic acids and are administered toanimals, then commercially available gene guns are a preferred methodfor delivery. This technique utilizes an instrument designed to propelDNA-coated gold particles directly into cells within the epidermis anddermis. DNA enters directly into dendritic cells, which leads to directpriming of CD8+ T cells. (Chen, 2000) In particular, gene gun deliveryby DNA coated gold beads may thus be preferable for use with compositionconstituents enhancing CD8+ T cell immune response for nucleic acidbased subunit compositions. (Chien-Fu Hung, 2003) Routes ofadministration for nucleic acid based compositions are summarized inFIG. 7 and below.

D. Description of Additional Alternative Embodiments and ImmuneStimulants

The immune response contemplated by the present invention may beenhanced by the use of non-specific or specific substances stimulatingimmune response. The present invention may be mixed with appropriateimmune stimulant or adjuvant, including those described as alternativeembodiments below. Such compositions may be used as appropriate for theapplication. Customary stimulants or adjuvant known in the art includeincomplete Freund's adjuvant, liposomes, etc. A preferred embodimentincludes one or more stimulant taken from customary adjuvants and/orthose compositions described further herein. In addition, DNA enhancescomplement activity and therefore, may be used concurrently as a DNAvaccine and an adjuvant. (The DPT vaccine is composed of three separatevaccine particles. The pertussis component acts as an adjuvant for theother two. (Parham, 2004) An analogous situation exists here, where aDNA vaccine (preferably encoding the sequence for the amino terminal endof the matrix protein (p17MA) and the covalent binding site formyristate on the HIV virus) for HIV disease would act as an adjuvant fora amino terminal end of the matrix protein (p17MA) and the covalentbinding site for myristate on the HIV virus subunit vaccine.)

To enhance immunogenicity of a recombinant bacterial or viral vectorsialic acid needs to be removed from the plasma membrane of the bacteriaor the protein coat and or envelope (if virus is enveloped) structure ofthe virus. Treatment with neuraminidase would effectively remove sialicacid residues without altering the protein structure of the bacteria orvirus.

In an alternative embodiment, the composition may be bound covalently orotherwise to polysaccharides composed of mannose or mannan. Binding orcoupling may be accomplished using methods known to those in the field.Mannose is a sugar found only on microorganisms and pathogens notordinarily found within the human body. Mannose binding protein (MBP) isa collectin, a C-type lectin that contains regions of collagenousstructure. It is present in normal human serum and consists of subunitseach composed of three polypeptide chains, forming a collagen-liketriple helix and three C-terminal globular carbohydrate recognitiondomains (CRDs). Six subunits together form an overall structureresembling the bouquet of tulip-like structure of C1q of the classicalcomplement pathway. Binding of MBP to carbohydrate initiates theclassical complement pathway to the activation of C1r₂ C1s₂. This mayresult in complement killing either directly through insertion of theterminal membrane attack complex or through opsonization by depositionof complement on the microbial surface. MBP may also activate C2 and C4via another newly described serine protease called MASP (1 and 2) serineproteases. Thus, MBP also exhibits complement independent opsonizingactivity, probably mediated by binding of the collagenous stalks to thecollectin receptor of phagocytic cells. (Presanis J. S., et al.,Biochemistry and Genetics of Mannan-binding Lectin (MBL), BiochemicalSociety Transactions, Vol. 31, pp 748-752 (2003) Any organism withmannose or mannan on its surface will stimulate the lectin pathway ofcomplement activation. A composition bound to such polysaccharides willbind with mannose binding lectin in the serum, activating the lectinpathway of the complement system. Thus, this alternative embodimentwould thereby enhance the overall immunologic response to the vaccine.

In another alternate embodiment, the composition may be combined withsubstances that stimulate or activate the alternative complementpathway. For example, it is known that certain forms of teichoic acidare potent activators of the alternative complement pathway.(Winkelstein J. A., J. of Immun., Vol. 120, pp 174-178 (1978)) Inaddition, zymosan, which may be derived from yeast cells, can inducecytokines and stimulate immune response in conjunction with thealternative pathway of the complement system. Zymosan is phagocytosed bymacrophages with or without opsonization, and therefore has a usefulimmunologic property of activating the alternative pathway ofcomplementation. The zymosan macrophage interaction is believed toenhance the Th-1 response. CD4 cells can be divided into Th-1 and Th-2cells. Th-1 cells activate cytotoxic T cells by producing IL-2; whereasTh-2 cells activate B-cells by producing primarily IL-4 and IL-5. Thelevel of Th-1 response produced by zymosan is regulated by C3 cleavagefragments, C3b and iC3b. The amplified C3b deposits on the acceptedsurface of zymosan and assembles macrophages, dendritic cells or otherantigen-presenting cells. Macrophages, dendritic cells, andantigen-presenting cells make an antigen presentation to Th-1 cellsafter opsonizing zymosan, and after antigen-specific macrophageactivation occurs. (Fearon D. T., et al., Proc. Natl. Acad. Sci, Vol.74, pp 1683-1687 (1977)) Zymosan can therefore be used as an adjuvant;it enhances both humoral and cell-mediated immune responses to HIVdisease. Thus, the composition may be bound covalently or otherwise tosubstances that stimulate the alternative complement pathway, such asteichoic acid or zymosan.

The adjuvant effect of zymosan on HIV specific DNA vaccine wasdemonstrated recently using a plasma vector (pCMV160 IIIb). Inlaboratory mice the plasmid vaccine was inoculated with and without thezymosan. Higher levels of both humoral immune response and HIV specificdelayed type hypersensitivity (DTH) response were observed when zymosanwas co-inoculated with the plasmid vector as to that using the plasmidvector alone. HIV specific cytotoxic T cell lymphocyte activity was alsoenhanced. The effects are suggested to be based on the consequences ofits (zymosan) recruitment and activation of macrophages, dendriticcells, or antigen-presenting cells through complement activation andespecially through the alternative pathway. These results suggestzymosan as an effective immunologic stimulant. (Ara, 2001)

Therefore, to enhance the immunogenicity of the composition, mannose,teichoic acid, zymosan, or some combination thereof may be bonded to theprotein component of the subunit vaccine. Preferably, thepolysaccharides will consist of sixteen separate saccharide units.(Pangburn, Michael K., Immun., Vol. 142, pp 2766-2770 (1989)) Thepreferred source for the carbohydrate/adjuvant component of the subunitvaccine would be the capsular polysaccharide of the yeast cell,Cryptococcus neoformans serotype C. (Sahu Arvind, et al., Biochem. J.,Vol 302, pp 429-436 (1994)) This yeast cell exhibits four branchingxylose sugars from each trimannose repeat unit. The thioester site ofthe C3 complement component demonstrates a strong preference for thisspecific carbohydrate sequence. This results in the cleavage of C3 intothe C3a fragment and C3b. The C3b molecule is a focal point in all threecomplement pathways.

Additionally, all glucose molecules and polysaccharides are to beremoved from the composition. The addition of insulin to a cell culturewill facilitate the transport of extracellular glucose across the plasmamembrane and into the cytoplasm of the cells. Free soluble glucosemolecules inhibit both the rate and the extent of C3b deposition. (SahuArvind, 1994)

In an alternate embodiment, the effect of heparin may be inhibited.Heparin is a cofactor necessary for effective Factor H function.(Maillet, Francoise, et al., Mol. Immun., Vol. 25, pp 917-923 (1988))(Maillet, Francoise, et al., Molecular Immun., Vol. 20, pp 1401-1404(1983)) Further, CypA uses heparin as a binding partner when binding tohost cells. (Saphire, Andrew C. S., et al., European Molecular Bio. J.18:6771-6785 (1999)) As noted above, Factor H is a major limitingprotein in the alternative complement pathway. The alternativecomplement pathway is the first arm of the immune system to respond tomicroorganisms or vaccines. Protamine binds heparin and is used toreduce the effective heparin in patients undergoing anticoagulation.(Furie, Bruce, Oral Anticoagulant Therapy, Hematology Basic Principles &Practice, Ch. 121 (3rd ed. 2000)) Recently, a less toxic heparinantagonist, low molecular weight protamine (LMWP) has become available.Protamine, or preferably LMWP for this embodiment, may be included as acomponent of the composition in order to impair the activity of Factor Hin limiting the alternative complement pathway. (Liang J. F, et al.,Biochemistry, Vol. 68, pp 116-120 (2002)) Alternatively, Heparinase isknown to degrade Heparin enzymatically.

Branched partially hydrolyzed polysaccharides of glucose known asdextrans have been used for effective plasma expanders. (Hoffman,Ronald, Hematology Basic Principles and Practice, 2177 (3rd ed. 2000))Dextran sulfate is a sodium salt of sulfuric acid esters of thepolysaccharide dextran. Soluble dextran sulfate with a molecular weightgreater than 5×10³ is an inducer of the alternative pathway ofcomplement. The number of sulfate groups per hundred glucose residues inthe dextran determined the activation potency of the dextran in thealternative pathway. The optimal degree of sulphation was 50-60 SO₄/100glucose molecules. (Burger, R., et al., Immunology, Vol. 29. pp 549-554(1975))

Sulphated sephadex (SS) is a cross-linked insoluble form of dextran.Like soluble dextran sulphate SS activate the alternative pathway ofcomplement and the classical pathway as well. Three variables controlthe activity of SS with both pathways of complement activity:

-   -   (1) Amount of sulphation.; the higher the sulphated content up        to 15.6% by weight resulted in higher complement activation. No        complement activation was noted with sulphate content less than        2.43%;    -   (2) Concentration of SS; higher concentrations result in        complement activation with a maximum C3 turnover at 40-50 μg/ml;        and    -   (3) Temperature; maximum C3 turnover was noted at 37° C. with a        total loss of activity at 4° C.        (Burger, R., et al., Immunology 33:827 (1977)) Both soluble and        insoluble forms of dextran (>5000 molecular weight) activate the        alternative pathway of complement. This is accomplished by        blocking the effect of factor H. (Burger, R., et al.,        European J. Immunology, pp. 291-295 (1981)) Low molecular weight        dextran sulfate (<5000) enhances factor H binding therefore it        limits the activity of the alternative pathway of complement.        (Seppo Meri, et. al., Proc. Natl. Acad. Sci, Vol 87, pp        3982-3986 (1990) DNA like heparin also increases factor H        binding. (Gardner, William D., Biochemical and Biophysical        Research Communications, Vol. 94, pp 61-67 (1980))

Therefore, to enhance immunogenicity dextran sulfate with a molecularweight >5000 with 50-60 SO₄/100 glucose molecules may be included in thecompound. Likewise SS with 15.6% SO₄ by weight at a concentration of40-50 μg/ml at a temperature of 37° would enhance the immunogenicity ofthe compound. Low molecular weight dextran would not be included in theformulation since it would increase factor H binding and decreasecomplement activation.

In a further alternate embodiment, substances that stabilize C3convertase may be used with the present invention. All three complementpathways lead to the production of C3b, which bonds covalently to thesurface of microorganisms or components of the microorganisms presentedin such an immunogenic composition. C3b is produced by enzymes known asC3 convertase. Cobra venom factor (CVF), derived from the snake Najakaouthia stabilizes this enzyme. (Alper, C. A., et al., Science, Vol.191, pp 1275-1276 (1976) The half life of CVF,C3b,Bb C3/C5 convertase isseven hours, in contrast to that of endogenously produced alternativecomplement pathway C3 convertase (C3b,Bb), which is 1.5 minutes. C3b,Bbis disassembled by Factor H and C3b is inactivated by the combinedaction of Factor H and Factor I. In contrast Factor CVF,C3b,Bb isresistant to all regulatory complement proteins. (Kock, Michael A., etal., J. of Biol. Chemistry, Vol. 279 pp 30836-30843 (2004)) C3b,Bbrequires additional C3b to act on C5 whereas CVF,Bb can cleave C5directly. Therefore, the CVF,Bb enzyme continuously activates C3 and C5.(Kock, 2004)

The biological function of CVF in cobra venom is believed to facilitatethe entry of the toxic venom components into the bloodstream. This isachieved by complement activation causing release of the anaphylatoxinsC3a, C5a and Bb, which increase vascular permeability. (Vogel, Carl W.,Immunoconjugates, Ch. 9 (1987)) CVF, despite its derivation from cobravenom, is a non-toxic protein; CVF can be isolated from the otherenzymes, polypeptides, etc., from cobra venom, which includes toxins.

Thus, administration of CVF results in an explosive production of C3b.(Vogel, 1987) (Kock, 2004) FIG. 8 illustrates the structural homologybetween C3 and CVF. C3b on the surface of microorganisms is recognizedby follicular dendritic cells within the lymph nodes as well as T cellsand B cells in the peripheral circulation and within the germinalcenters of the lymph nodes. C3b is a powerful opsonin. Opsonins triggerseveral arms of the immune system simultaneously. (Hoffman, Ronald,Hematology Basic Principles and Practice, Ch. 27 (3rd ed. 2000)) Thus,in an alternative embodiment, CVF may be used as a component of thecomposition.

The preferred form of CVF is dCVF (De-α-galactosylated CVF). (Gowda, D.C., et al., “Immunoreactivity and function of Oligosaccharides in CobraVenom Factor,” J. of Immun., pp. 2977-2986, (Dec. 21, 1993)) Naturallyoccurring CVF is characterized by an unusual polysaccharide which is afucosylated biantennary complex-type N-linked chain containing anα-galactosylated Lex antigenic epitope, Galα-3Galβ1-4 (Fucal-3)GlcNAcβ1. Removal of this polysaccharide can be accomplished byincubating CVF with peptide-N-glycosidase F (N-glycanase) at 37° C. for18 to 23 hours at a ph of 8.0. Removal of this novel polysaccharide fromCVF is necessary since 1% of human IgG reacts with the terminalGalα1-3Galβ1 sequence of CVF. However removal of this polysaccharidedoes not interfere with the complement fixation character of themolecule nor does it result in a shorter half life of the molecule. dCVFwill be covalently bound to the polysaccharide unit(s) comprising theimmunogenic composition.

In another embodiment, nickel compounds may be added to the composition.It has been shown that nickel is effective in enhancing the C3convertase activity of both the classic and the alternative complementpathways. (Fishelson, Z., et al., J. of Immun., Vol. 129, pp 2603-2607(1982)) Average nickel intake for average adults is estimated to be 60to 260 micrograms per day, with an environmental health reference doseof 0.02 milligram per kilogram body weight per day (mg/kg/d). (U.S. EPA,1986) It is contemplated that the present invention would include Nickelpreferable in the form of nickel chloride on the order of average dailyintake well below the reference dose. Therefore, the present inventionmay be produced using nickel to enhance immune response.

E. Summary

To prepare the composition that constitutes the vaccine agent for theinvention, it is possible to use known methods of purification,synthesis, or genetic engineering. Practitioners skilled in the art mayisolate and purify a fragment, or prepare a sequence encoding the aminoterminal end of the matrix protein (p17MA) and the covalent binding sitefor myristate on the HIV virus. Protein fragments, naked DNA/RNA,recombinant DNA/RNA, or messenger RNA may be incorporated intopharmaceutical compositions appropriate for the anticipated method ofadministration, such as carriers or excipients. An animal or subject forwhich an immune response according to the present invention is desiredmay be administered the composition; a therapeutically effective dosewould be that amount necessary to reverse specific immune suppression,to the extent desired, and determined using standard means, such asChromium Release Assay, Intracellular Cytokine Assay,Lympho-proliferative Assay (LPA), Interferon Gamma (IFN-gamma) ELISpotAssays, and MHC Tetramer Binding Assays. The MHC Tetramer Binding Assayis preferable. These same laboratory tests would be applied to measurethe immune response of an uninfected subject.

The analysis and development of the immunogenic composition shouldincorporate a wide range of doses of inactivated particulate forevaluation. Animal trials should consider differences in size, species,and immunological characteristics; it is anticipated that immunologicaldifferences between humans and animals may relegate animal trials totoxicity analysis. Clinical trials will involve at least the standardthree phase model, ranging from safety and dosage in a small population,safety and immunogenicity in a second phase of several hundredvolunteers, to a large scale effectiveness phase. The clinical trialsshould include appropriate exclusionary criteria as is customary, suchas exclusion for other immune suppression conditions, pregnancy, activedrug use, etc. A starting dose for trials with subunit proteins may be10 micrograms/strain for juveniles and 20 micrograms/strain for adults.For naked DNA vaccines a starting dose of 1 microgram/strain for allages would be appropriate.

Administration may be made in a variety of routes, for example orally,transbucally, transmucosally, sublingually, nasally, rectally,vaginally, intraocularly, intramuscularly, intralymphatically,intravenously, subcutaneously, transdermally, intradermally, intratumor, topically, transpulmonarily, by inhalation, by injection, or byimplantation, etc. Various forms of the composition may include, withoutlimitation, capsule, gel cap, tablet, enteric capsule, encapsulatedparticle, powder, suppository, injection, ointment, cream, implant,patch, liquid, inhalant, or spray, systemic, topical, or other oralmedia, solutions, suspensions, infusion, etc. Because some of the firsttargets for infection with HIV are epithelial cells and Langerhans cellsin the skin and rectal and vaginal mucosa, then a preferable embodimentof delivery is dermal combined with rectal and/or vaginal suppositories.HIV is contracted predominantly by rectal and vaginal intercourse.Therefore rectal and/or vaginal suppository administration of thevaccine would be the preferred administration methodology. In addition,the present invention may be combined with other therapeutic agents,such as cytokines, including natural, recombinant and mutated forms,fragments, fusion proteins, and other analogues and derivatives of thecytokines, mixtures, other biologically active agents and formulationadditives, etc. Those skilled in the art will recognize that forinjection, formulation in aqueous solutions, such as Ringer's solutionor a saline buffer may be appropriate. Liposomes, emulsions, andsolvents are other examples of delivery vehicles. Oral administrationwould require carriers suitable for capsules, tablets, liquids, pills,etc, such as sucrose, cellulose, etc.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention.

1. A composition for eliciting an immune response to HIV comprising, ina pharmaceutically acceptable support, an effective quantity of at leastone covalent binding site of myristate on the HIV matrix protein.
 2. Acomposition according to claim 1, in which said binding site isexpressed by a recombinant carrier.
 3. A composition according to claim2, wherein said recombinant carrier is a virus.
 4. A compositionaccording to claim 3, wherein said virus is a herpes virus.
 5. Acomposition according to claim 4, wherein said herpes virus is EpsteinBarr virus.
 6. A composition according to claim 3, wherein said virus isa poliovirus.
 7. A composition according to claim 3, wherein saidcomposition has been treated with neuraminidase, trypsin, or otherappropriate enzyme to remove sialic acid.
 8. A composition according toclaim 2, wherein said recombinant carrier is bacteria.
 9. A compositionaccording to claim 8, wherein said bacteria is Bacillus Calmette-Guerin.10. A composition according to claim 8, wherein said bacteria isListeria monocytogenes.
 11. A composition according to claim 8, whereinsaid composition has been treated with neuraminidase, trypsin, or otherappropriate enzyme to remove sialic acid.
 12. A composition according toclaim 2, wherein said recombinant carrier is yeast.
 13. A compositionaccording to claim 12, wherein said yeast is Saccharomyces cerevisiae.14. A composition according to claim 1, in which said binding site isexpressed by messenger RNA.
 15. Use of the composition according toclaim 1 for preparation of a medication for eliciting an immune responseto HIV.
 16. A method of eliciting an immune response in an animal, whichcomprises administering a composition comprising, in a pharmaceuticallyacceptable support, an effective quantity of at least one covalentbinding site of myristate on the HIV matrix protein.
 17. A methodaccording to claim 16, wherein the composition is administered, orally,transbucally, transmucosally, sublingually, nasally, rectally,vaginally, intraocularly, intramuscularly, intralymphatically,intravenously, subcutaneously, transdermally, intradermally, intratumor, topically, transpulmonarily, by inhalation, by injection, or byimplantation.
 18. A method according to claim 16, wherein thecomposition is administered, by capsule, gelcap, tablet, entericcapsule, encapsulated particle, powder, suppository, injection,ointment, cream, implant, patch, liquid, inhalant, or spray.
 19. Acomposition according to claim 1, wherein said composition is combinedwith an immune stimulant.
 20. A composition according to claim 19,wherein said immune stimulant is an adjuvant.
 21. A compositionaccording to claim 19, wherein said immune stimulant comprisespolysaccharides composed of at least one mannose in a form capable ofbinding to said composition.
 22. A composition according to claim 19,wherein said immune stimulant comprises teichoic acid in a form capableof binding to said composition.
 23. A composition according to claim 19,wherein said immune stimulant comprises zymosan in a form capable ofbinding to said composition.
 24. A composition according to claim 19,wherein said immune stimulant comprises cryptococcus neoformans serotypeC having a polysaccharide capsule capable of binding to saidcomposition.
 25. A composition according to claim 19, wherein saidimmune stimulant comprises protamine in a form capable of binding toheparin.
 26. A composition according to claim 19, wherein said immunestimulant comprises a heparinase.
 27. A composition according to claim19, wherein said immune stimulant comprises cobra venom factor in a formadapted to enhance production of C3b.
 28. A composition according toclaim 27, wherein said cobra venom factor is dCVF.
 29. A compositionaccording to claim 19, wherein said immune stimulant comprises Nickel ina form adapted to enhance C3 convertase activity.
 30. A compositionaccording to claim 19, wherein said immune stimulant comprises sulfatedpolyanions capable of absorbing Factor H.
 31. A composition according toclaim 1, wherein polyanions within the composition capable ofpotentiating Factor H are substantially removed from the composition.32. A composition for eliciting an immune response to HIV comprising, ina pharmaceutically acceptable support, an effective quantity ofGARASVLSSGG peptide of the HIV matrix protein.